Previous studies suggested that tyrosine kinase activation is an important signal transduction event in the IL-1 response of chondrocytes. The present study identifies the mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase (ERK)-1 and ERK-2 as major tyrosine phosphorylated proteins in IL-1 stimulated chondrocytes. Kinase assays on immunoprecipitates with myelin basic protein as substrate showed that ERK-1 and ERK-2 activation was detectable within 5 min after IL-1 stimulation and decreased to baseline within 60 min. Analysis of other members of the MAP kinase family showed that chondrocytes also express c-Jun NH2 terminal kinase (JNK)-1, JNK-2, and p38 proteins. These kinases were time-dependently activated by IL-1. Among other chondrocyte activators tested, only TNF activated all three of the MAP kinase subgroups. JNK and p38 were not activated by any of the other cytokines and growth factors tested. However, ERK was also activated by PDGF, IGF-1, and IL-6. Phorbol 12-myristate 13-acetate, calcium ionophore, and cAMP analogues only increased ERK activity but had no significant effects on JNK or p38. These results suggest differential activation of MAP kinase subgroups by extracellular stimuli. ERK is activated in response to qualitatively diverse extracellular stimuli and various second messenger agonists. In contrast, JNK and p38 are only activated by IL-1 or TNF, suggesting that these kinases participate in the induction of the catabolic program in cartilage.

NM23-H2/NDP kinase B has been identified as a sequence-specific DNA-binding protein with affinity for a nuclease-hypersensitive element of the c-MYC gene promoter (Postel et al., 1993). The ability of Nm23-H2 to activate c-MYC transcription in vitro and in vivo via the same element demonstrates the biological significance of this interaction. Mutational analyses have identified Arg34, Asn69 and Lys135 as critical for DNA binding, but not required for the NDP kinase reaction. However, the catalytically important His118 residue is dispensible for sequence-specific DNA binding, suggesting that sequence-specific DNA recognition and phosphoryl transfer are independent properties. Nm23-H2 also has an activity that cleaves DNA site-specifically, involving a covalent protein-DNA complex. In a DNA sequence-dependent manner, Nm23-H2 recognizes additional target genes for activation, including myeloperoxidase, CD11b, and CCR5, all involved in myeloid-specific differentiation. Moreover, both NM23-H1 and Nm23-H2 bind to nuclease hypersensitive elements in the platelet-derived growth factor PDGF-A gene promoter sequence-specifically, correlating with either positive or negative transcriptional regulation. These data support a model in which NM23/NDP kinase modulates gene expression through DNA binding and subsequent structural transactions.

Upon stimulation by its ligand, the platelet-derived growth factor (PDGF) receptor associates with the 85-kDa subunit of phosphatidylinositol (PI) 3-kinase. The 85-kDa protein (p85) contains two Src homology 2 (SH2) domains and one SH3 domain. To define the part of p85 that interacts with the PDGF receptor, a series of truncated p85 mutants was analyzed for association with immobilized PDGF receptor in vitro. We found that a fragment of p85 that contains a single Src homology domain, the C-terminal SH2 domain (SH2-C), was sufficient for directing the high-affinity interaction with the receptor. Half-maximal binding of SH2-C to the receptor was observed at an SH2-C concentration of 0.06 nM. SH2-C, like full-length p85, was able to distinguish between wild-type PDGF receptor and a mutant receptor lacking the PI 3-kinase binding site. An excess of SH2-C blocked binding of full-length p85 and PI 3-kinase to the receptor but did not interfere with the binding of two other SH2-containing proteins, phospholipase C-gamma and GTPase-activating protein. These results demonstrate that a region of p85 containing a single SH2 domain accounts both for the high affinity and specificity of binding of PI 3-kinase to the PDGF receptor.

OBJECTIVE

We analyzed the expression of platelet-derived growth factor D (PDGF-D) in an experimental bile duct-ligated (BDL) rat model and assessed its biological function in cultured hepatic stellate cells (HSC) and myofibroblasts (MFB).

METHODS

The mRNA for PDGF-A, -B, -C, -D and for PDGF receptor-alpha and -beta chains (PDGFRalpha and PDGFRbeta) in normal and fibrotic rat livers was assessed quantitatively. Protein levels of PDGF-D were quantified by immunoblotting and immunohistochemistry.

RESULTS

The relative mRNA expression of all PDGF isoforms and receptors upregulated upon BDL and PDGF-A, -B and -D expression was significantly higher than that of PDGF-C. PDGF-D and PDGFRbeta protein also increased markedly. Immunostaining revealed that PDGF-D is localized along the fibrotic septa of the periportal- and perisinusoidal areas. Besides PDGF-B, PDGF-D is the second most potent PDGF isoform in PDGFRbeta signaling within HSC/MFB, evidenced by PDGFRbeta autophosphorylation and activation of the downstream signaling molecules ERK1/2-, JNK-, p38 MAPK, and PKB/Akt while PDGF-C effects were minimal. PDGF-D exerted mitogenic and fibrogenic effects in both cultured HSC and MFB comparable to PDGF-B but PDGF-A and -C showed only marginal fibrogenic effects.

CONCLUSIONS

PDGF-D possesses potential pathogenetic properties for HSC activation and matrix remodeling in liver fibrosis.

Iba1 is a macrophage/microglia-specific calcium-binding protein that is involved in RacGTPase-dependent membrane ruffling and phagocytosis. In this study, we introduced Iba1 into Swiss 3T3 fibroblasts and demonstrated the enhancement of platelet-derived growth factor (PDGF)-induced membrane ruffling and chemotaxis. Wortmannin treatment did not completely suppressed this enhanced membrane ruffling in Iba1-expressing cells, whereas it did in Iba1-nonexpressing cells, suggesting that the enhancement is mediated through a phosphatidylinositol 3-kinase (PI3K)-independent signaling pathway. Porcine aorta endothelial cells transfected with expression constructs of Iba1 and PDGF receptor add-back mutants were used to analyze the signaling pathway responsible for the Iba1-induced enhancement of membrane ruffling. In the absence of Iba1 expression, PDGF did not induced membrane ruffling in cells expressing the Tyr-1021 receptor mutant, which is capable of activating phospholipase C-gamma (PLC-gamma) but not PI3K. In contrast, in the presence of Iba1 expression, membrane ruffling was formed in cells expressing the Tyr-1021 mutant. In addition, Rac was shown to be activated during membrane ruffling in cells expressing Iba1 and the Tyr-1021 mutant. Furthermore, dominant negative forms of PLC-gamma completely suppressed PDGF-induced Iba1-dependent membrane ruffling and Rac activation. These results indicate the existence of a novel signaling pathway where PLC-gamma activates Rac in a manner dependent on Iba1.

In cultured vascular smooth muscle cells (VSMC), the vasculotrophic factor, angiotensin II (AngII) activates three major MAPKs via the G(q)-coupled AT1 receptor. Extracellular signal-regulated kinase (ERK) activation by AngII requires Ca(2+)-dependent "transactivation" of the EGF receptor that may involve a metalloprotease to stimulate processing of an EGF receptor ligand from its precursor. Whether EGF receptor transactivation also contributes to activation of other members of MAPKs such as p38MAPK and c-Jun N-terminal kinase (JNK) by AngII remains unclear. In the present study, we have examined the effects of a synthetic metalloprotease inhibitor BB2116, and the EGF receptor kinase inhibitor AG1478 on AngII-induced activation of MAPKs in cultured VSMC. BB2116 markedly inhibited ERK activation induced by AngII or the Ca(2+) ionophore without affecting the activation by EGF or PDGF. BB2116 as well as HB-EGF neutralizing antibody inhibited the EGF receptor transactivation by AngII, suggesting a critical role of HB-EGF in the metalloprotease-dependent EGF receptor transactivation. In addition to the ERK activation, activation of p38MAPK and JNK by AngII was inhibited by an AT1 receptor antagonist, RNH6270. and EGF markedly activate p38MAPK, whereas but not EGF markedly activates JNK, indicating the possible contribution of the EGF receptor transactivation to the p38MAPK activation. The findings that both BB2116 and AG1478 specifically inhibited activation of p38MAPK but not JNK by AngII support this hypothesis. From these data, we conclude that ERK and p38MAPK activation by AngII requires the metalloprotease-dependent EGF receptor transactivation, whereas the JNK activation is regulated without involvement of EGF receptor transactivation.

One of the early events after stimulation of Swiss 3T3 cells with either platelet-derived growth factor (PDGF), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), diacylglycerol, or several other mitogens is the near stoichiometric phosphorylation at tyrosine and serine of a scarce cytoplasmic protein (p42). TPA and diacylglycerol are known to directly stimulate the activity of a protein-serine/threonine kinase, protein kinase C (PKC). PDGF and several other mitogens stimulate tyrosine kinases directly and PKC indirectly. We have therefore examined the involvement of PKC in p42 tyrosine phosphorylation in Swiss 3T3 cells. Firstly, six agents which stimulated phosphorylation of p42 also stimulated phosphorylation of a known PKC substrate, an 80,000-Mr protein (p80). Secondly, in PKC-deficient cells (cells in which PKC activity was reduced to undetectable levels by prolonged exposure to TPA), PDGF-induced p42 phosphorylation was reduced three- to fourfold. Phosphoamino acid analysis of phosphorylated p42 from PDGF-stimulated PKC-deficient cells revealed primarily phosphoserine and only a trace of phosphotyrosine, suggesting that the reduction in PDGF-stimulated tyrosine phosphorylation of p42 resulting from PKC deficiency is greater than three- to fourfold. Finally, comparison of antiphosphotyrosine immunoprecipitates of PKC-deficient versus naive cells revealed that most other PDGF-induced tyrosine phosphorylation events were quite similar. These data suggest that mitogens such as PDGF, which directly stimulate phosphorylation of some proteins at tyrosine, induce p42 tyrosine phosphorylation via a cascade of events involving PKC.

Adult rat arterial smooth muscle cells (SMC) in primary culture modulate from contractile to synthetic phenotype. This process includes partial loss of myofilaments and formation of an extensive rough endoplasmic reticulum and a large Golgi complex. It gives the cells the ability to initiate DNA synthesis and actively proliferate when stimulated with serum or isolated growth factors. After a few divisions, growth becomes partly independent of exogenous mitogens and does not cease until multiple cell layers have been formed. Here, it is demonstrated that serum-free conditioned medium from primary cultures of adult rat arterial SMC contains a factor that initiates DNA synthesis in growth-arrested secondary cultures of SMC. The mitogenic activity was neutralized by antibodies to platelet-derived growth factor (PDGF), and no mitogenic activity occurred in conditioned medium from cultures pretreated with actinomycin D, excluding release into the medium of PDGF adsorbed to the plastic vessels during the initial culture in serum-containing medium. Exposure of human fibroblasts to samples of the conditioned medium at 4 degrees C inhibited subsequent binding of 125I-labeled PDGF. It was further shown that the SMC of the primary cultures were able to initiate DNA synthesis in a chemically defined medium lacking PDGF and other growth factors. During the early, most active, and partly autonomous growth phase, the SMC had a low binding capacity for 125I-labeled PDGF and responded but little to stimulation with exogenous PDGF. Later on, with increasing cell density and decreasing growth rate, the ability to bind and respond to exogenous PDGF increased. Taken together, the observations suggest that modulation of SMC from contractile to synthetic phenotype is accompanied by production of a PDGF-like protein and autocrine or possibly by mitogen-independent initiation of DNA synthesis. Functionally, this may be important during wound healing and in the development of atherosclerotic lesions.

The mechanism of leukaemogenic transformation by human T-cell leukaemia/lymphoma virus (HTLV), a retrovirus implicated in the aetiology of certain adult T-cell leukaemias and lymphomas, is unknown but is conceivably associated with the expression of the cellular analogues of retroviral oncogenes. The HUT-102 cell line, derived from a cutaneous T-cell lymphoma and infected with HTLV, expresses several cellular oncogenes. It is unusual among haemopoietic cell lines in that one of these is c-sis, the gene from which the oncogene v-sis of the simian sarcoma virus was derived, and perhaps the gene for platelet-derived growth factor (PDGF). To explore the possible role of c-sis expression in HTLV-induced disease, we have obtained cDNA clones of c-sis from HUT-102 cells. Here we describe two such clones and report that one of them transforms NIH-3T3 cells. This is the first example of transformation of NIH-3T3 cells by a human onc gene other than c-ras or Blym, as well as the first demonstration of transformation by a human cDNA clone.

Oxidative stress is known to induce cell death in a wide variety of cell types, apparently by modulating intracellular signaling pathways. In this study, we have examined the activation of mitogen-activated protein kinase (MAPK) cascades in relation to oxidant-induced cell death in an oligodendrocyte cell line, central glia-4 (CG4). Exposure of CG4 cells to hydrogen peroxide (H2O2) resulted in an increased tyrosine phosphorylation of several protein species, including the abundantly expressed platelet-derived growth factor (PDGF) receptor and the activation of the three MAPK subgroups, i.e., extracellular signal-regulated kinase (ERK), p38 MAPK, and c-Jun N-terminal kinase (JNK). Dose-response studies showed differential sensitivities of PDGF receptor phosphorylation (>1 mM) and ERK/p38 MAPK (>0.5 mM) and JNK (>0.1 mM) activation by H2O2. The activation of ERK was inhibited by PD98059, a specific inhibitor of the upstream kinase, MAPK or ERK kinase (MEK). H2O2 also activated MAPK-activated protein kinase-2, and this activation was blocked by SB203580, a specific inhibitor of p38 MAPK. The oxidant-induced cell death was indicated by morphological changes, decreased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction, and DNA fragmentation. These effects were suppressed dose-dependently by the MEK inhibitor PD98059. The results demonstrate that H2O2 induces the activation of multiple MAPKs in oligodendrocyte progenitors and that the activation of ERK is associated with oxidant-mediated cytotoxicity.

A hallmark of smooth muscle cell (SMC) phenotypic modulation in atherosclerosis and restenosis is suppression of SMC differentiation marker genes, proliferation, and migration. Blockade of intermediate-conductance Ca(2+)-activated K(+) channels (IKCa1) has been shown to inhibit restenosis after carotid balloon injury in the rat; however, whether IKCa1 plays a role in SMC phenotypic modulation is unknown. Our objective was to determine the role of IKCa1 channels in regulating coronary SMC phenotypic modulation and migration. In cultured porcine coronary SMCs, platelet-derived growth factor-BB (PDGF-BB) increased TRAM-34 (a specific IKCa1 inhibitor)-sensitive K(+) current 20-fold; increased IKCa1 promoter histone acetylation and c-jun binding; increased IKCa1 mRNA approximately 4-fold; and potently decreased expression of the smooth muscle differentiation marker genes smooth muscle myosin heavy chain (SMMHC), smooth muscle alpha-actin (SMalphaA), and smoothelin-B, as well as myocardin. Importantly, TRAM-34 completely blocked PDGF-BB-induced suppression of SMMHC, SMalphaA, smoothelin-B, and myocardin and inhibited PDGF-BB-stimulated migration by approximately 50%. Similar to TRAM-34, knockdown of endogenous IKCa1 with siRNA also prevented the PDGF-BB-induced increase in IKCa1 and decrease in SMMHC mRNA. In coronary arteries from high fat/high cholesterol-fed swine demonstrating signs of early atherosclerosis, IKCa1 expression was 22-fold higher and SMMHC, smoothelin-B, and myocardin expression significantly reduced in proliferating vs. nonproliferating medial cells. Our findings demonstrate that functional upregulation of IKCa1 is required for PDGF-BB-induced coronary SMC phenotypic modulation and migration and support a similar role for IKCa1 in coronary SMC during early coronary atherosclerosis.

Binding of platelet-derived growth factor (PDGF) to the PDGF receptor (PDGFR) beta subunit triggers receptor tyrosine phosphorylation and the stable association of a number of signal transduction molecules, including phospholipase C gamma (PLC gamma), the GTPase activating protein of ras (GAP), and phosphatidylinositol-3 kinase (PI3K). Previous reports have identified three PDGFR tyrosine phosphorylation sites in the kinase insert domain that are important for stable association of GAP and PI3K. Two of them, tyrosine (Y) 740, and Y-751 are required for the stable association of PI3K, while Y-771 is required for binding of GAP. Here we present data for two additional tyrosine phosphorylation sites, Y-1009 and Y-1021, that are both in the carboxy-terminal region of the PDGFR. Characterization of PDGFR mutants in which these phosphorylation sites are substituted with phenylalanine (F) indicated that Y-1021 and Y-1009 were required for the stable association of PLC gamma and a 64-kDa protein, respectively. An F-1009/F-1021 double mutant selectively failed to bind both PLC gamma and the 64-kDa protein, whereas all of the carboxy-terminal mutants bound wild-type levels of GAP and PI3K. The carboxy terminus encodes the complete binding site for PLC gamma, since a phosphorylated carboxy-terminal fusion protein selectively bound PLC gamma. To determine the biological consequences of failure to associate with PLC gamma, we measured PDGF-dependent inositol phosphate production and initiation of DNA synthesis. The PDGFR mutants that failed to associate with PLC gamma were not able to mediate the PDGF-dependent production of inositol phosphates. Since tyrosine phosphorylation of PLC gamma enhances its enzymatic activity, we speculated that PDGFR mutants that failed to activate PLC gamma were unable to mediate its tyrosine phosphorylation. Surprisingly, the F-1021 receptor mediated readily detectable levels of PDGF-dependent PLC gamma tyrosine phosphorylation. Thus, the production of inositol phosphates requires not only PLC gamma tyrosine phosphorylation but also its association with the PDGFR. Comparison of the mutant PDGFRs' abilities to initiate PDGF-dependent DNA synthesis indicated that failure to associate with PLC gamma and produce inositol phosphates diminished the mitogenic response by 30%. In contrast, preventing the PDGFR from binding the 64-kDa protein did not compromise PDGF-triggered DNA synthesis at saturating concentrations of PDGF. Thus, it appears that phosphorylation of the PDGFR at Y-1021 is required for the stable association of PLC gamma to the receptor's carboxy terminus, the production of inositol phosphates, and initiation of the maximal mitogenic response.

Activation of growth factor receptor protein tyrosine kinases frequently results in the binding of numerous proteins to their tyrosine-phosphorylated cytoplasmic domains. These interactions involve the SH2 domains of the binding proteins and phosphorylated tyrosines on the receptor molecules, with the specificity of interaction dictated by the amino acid composition surrounding the phosphorylated tyrosine. In the case of the platelet-derived growth factor (PDGF) receptor, the major binding site for Src family tyrosine kinases is in the juxtamembrane domain and includes tyrosine 579 (Mori, S., Rönnstrand, L., Yokote, K., Engström, A., Courtneidge, S. A., Claesson-Welsh, L., and Heldin, C-H. (1993) EMBO J. 12, 2257-2264). To analyze in more detail which amino acids surrounding the phosphorylated tyrosine at position 579 were important for high affinity interaction with Src family kinases, we synthesized a series of phosphopeptides corresponding to this binding site in which single amino acids were individually changed and tested their ability to compete with the PDGF receptor for binding of Fyn. We found that not only the three residues carboxyl-terminal to the phosphorylated tyrosine were important but that also residues at positions -1 and +4 relative to the tyrosine were required. Phosphorylation of both tyrosines 579 and 581 significantly increased competition efficiency. The activated colony stimulating factor-1 (CSF-1) receptor, which is known to associate with Src family kinases, has a sequence in its juxtamembrane region similar to that surrounding Tyr-579 of the PDGF receptor, and a phosphopeptide modeled on this sequence competed the association of Fyn with the receptor in vitro. Furthermore, mutational analysis demonstrated that these sequences were required for the efficient association of Src family kinases with the activated CSF-1 receptor in vivo. Phosphopeptides corresponding to the Src family binding sites of both PDGF and CSF-1 receptors activated Src kinase activity in vitro. These observations support a model in which the enzymatic activity of Src family tyrosine kinases is controlled by intra- and intermolecular interactions of tyrosine-phosphorylated peptides with the SH2 domain of the kinases.

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.

OBJECTIVE

The aim of this study was to evaluate and compare the clinical and inflammatory parameters with the addition of platelet-derived growth factor (PDGF) in primary anterior cruciate ligament (ACL) reconstruction with bone-patellar tendon-bone allograft.

METHODS

We prospectively randomized 100 patients undergoing arthroscopic patellar tendon allograft ACL reconstruction to a group in whom platelet-enriched gel was used (n = 50) and a non-gel group (n = 50). The platelet concentration was 837 x 10(3)/mm(3), and the gel was introduced inside the graft and the tibial tunnel. Demographic data were comparable between groups. The mean follow-up was 24 months for both groups and included a history, clinical evaluation with the International Knee Documentation Committee score, radiographs, and magnetic resonance imaging.

RESULTS

There were no differences in the number of associated injuries. The results did not show any statistically significant differences between the groups for inflammatory parameters (perimeters of the knee and C-reactive protein level), magnetic resonance imaging appearance of the graft, and clinical evaluation scores (visual analog scale, International Knee Documentation Committee, and KT-1000 arthrometer [MEDmetric, San Diego, CA]).

CONCLUSIONS

At this time, the therapeutic role of PDGF in ACL reconstruction remains unclear. The use of PDGF, on the graft and inside the tibial tunnel, in patients treated with bone-patellar tendon-bone allografts has no discernable clinical or biomechanical effect at 2 years' follow-up. More clinical studies will be needed to show the efficacy and use of these factors in daily practice in ACL reconstruction.

METHODS

Level I, prospective, randomized, double-blind study.

OBJECTIVE

Investigations on the combination of radiotherapy with vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) antiangiogenic agents, which has the potential to improve the clinical outcome in cancer patients.

METHODS

Here, we analyze the combined VEGF (SU5416) and PDGF (SU6668) receptor tyrosine kinase inhibition with irradiation in human endothelium (HUVEC), prostate cancer (PC3), and glioblastoma (U87) in vitro and in vivo.

RESULTS

Combined inhibition of VEGF and PDGF signaling resulted in enhanced apoptosis, reduced cell proliferation, and clonogenic survival as well as reduced endothelial cell migration and tube formation compared with single pathway inhibition. These effects were further enhanced by additional irradiation. Likewise, in PC3 and U87 tumors growing s.c. on BALB/c nu/nu mice, dual inhibition of VEGF and PDGF signaling significantly increased tumor growth delay versus each monotherapy. Interestingly, radiation at approximately 20% of the dose necessary to induce local tumor control exerts similar tumor growth-inhibitory effects as the antiangiogenic drugs given at their maximum effective dose. Addition of radiotherapy to both mono- as well as dual-antiangiogenic treatment markedly increased tumor growth delay. With respect to tumor angiogenesis, radiation further decreased microvessel density (CD31 count) and tumor cell proliferation (Ki-67 index) in all drug-treated groups. Of note, the slowly growing PC3 tumor responded better to the antiangiogenic drug treatments than the faster-growing U87 tumor. In addition to the beneficial effect of abrogating VEGF survival signaling when combined with radiation, we identified here a novel mechanism for the tumor escape from radiation damage. We found that radiation induced up-regulation of all four isoforms of PDGF (A-D) in endothelial cells supporting adjacent smooth muscle cells resulting in a prosurvival effect of radiation. The addition of SU6668 attenuated this undesirable paracrine radiation effect, which may rationalize the combined application of radiation with PDGF signaling inhibition to increase antitumor effects.

CONCLUSIONS

A relative low radiation dose markedly enhances local antitumor effects of combined VEGF and PDGF signaling inhibition, suggesting a promising combination regimen for local tumor treatment with radiotherapy remaining an essential element.

Central nervous system development requires precise and localized regulation of neural precursor behaviour. Here we show how the interaction between growth factor and integrin signalling pathways provides a mechanism for such precision in oligodendrocyte progenitor (OP) proliferation. While physiological concentrations of platelet-derived growth factor (PDGF) were not in themselves sufficient to promote OP proliferation, they did so on extracellular matrix (ECM) substrates that bind alpha(v)beta3 integrin. Upon PDGF-AA exposure and alpha(v)beta3 engagement, a physical co-association between both receptors was demonstrated, confirming the interaction between these signalling pathways. Furthermore, we found that PDGFalphaR stimulated a protein kinase C-dependent activation of integrin alpha(v)beta3, which in turn induced OP proliferation via a phosphatidylinositol 3-kinase-dependent signalling pathway. These studies establish a mechanism by which OP proliferation is dependent on the availability of both an ECM ligand and a mitogenic growth factor. Growth factor- mediated integrin activation is the critical integrative step in proliferation signalling, and ensures that the response of neural precursor cells to long-range cues can be regulated by their cellular neighbours, allowing precise control of cell behaviour during development.

The human sis proto-oncogene contains the coding sequence for one of two polypeptide chains present in preparations of biologically active human platelet-derived growth factor (PDGF). A human clone, c-sis clone 8, which contains all of the v-sis-related sequences present in human DNA, was transcriptionally inactive when transfected into NIH/3T3 cells. When placed under the control of a retrovirus LTR, the clone was transcribed at levels comparable to that observed in cells transformed by SSV DNA. However, c-sis clone 8 DNA did not express detectable sis/PDGF-2 proteins and lacked biologic activity. A putative upstream exon was identified by its ability to detect the 4.2 kb sis-related transcript in certain human cells. When this sequence was inserted in the proper orientation between the LTR and c-sis clone 8, the chimeric molecule acquired high titered transforming activity, comparable to that of SSV DNA. Transformants containing this construct expressed human sis/PDGF-2 translational products. Thus the normal coding sequence for a human growth factor has transforming activity when expressed in an appropriate assay cell.

Polypeptide growth factors, such as platelet-derived growth factor (PDGF), promote the reinitiation of DNA synthesis and cell growth through multiple intracellular signaling pathways that converge in the nucleus to regulate the activity of transcription factors, thereby controlling the expression of growth-promoting genes. Among them, the AP-1 (activating protein-1) family of transcription factors, including c-Fos and c-Jun family members, plays a key role, as AP-1 activity is potently activated by PDGF and is required to stimulate cell proliferation. However, the nature of the pathways connecting PDGF receptors to AP-1 is still poorly defined. In this study, we show that PDGF regulates AP-1 by stimulating the expression and function of c-Fos through extracellular signal-regulated kinase (ERK). The latter involves the direct phosphorylation by ERK of multiple residues in the carboxyl-terminal transactivation domain of c-Fos, which results in its increased transcriptional activity. Interestingly, the phosphorylation of c-Fos by ERK was required for the ability of PDGF and serum to stimulate the activity of c-Fos as well as AP-1-dependent transcription. Furthermore, we provide evidence that the ERK-dependent activation of c-Fos is an integral component of the mitogenic pathway by which PDGF regulates normal and aberrant cell growth.

The mechanism by which the ubiquitously expressed Src family kinases regulate mitogenesis is not well understood. Here we report that cytoplasmic tyrosine kinase c-Abl is an important effector of c-Src for PDGF- and serum-induced DNA synthesis. Inactivation of cytoplasmic c-Abl by the kinase-inactive Abl-PP-K(-) (AblP242E/P249E/K290M) or by microinjection of Abl neutralizing antibodies inhibited mitogenesis. The kinase-inactive SrcK295M induced a G(1) block that was overcome by the constitutively active Abl-PP (AblP242E/P249E). Conversely, the inhibitory effect of Abl-PP-K(-) was not compensated by Src. c-Src-induced c-Abl activation involves phosphorylation of Y245 and Y412, two residues required for c-Abl mitogenic function. Finally, we found that p53 inactivation and c-myc expression, two cell cycle events regulated by Src during mitogenesis, also implied c-Abl: c-Abl function was dispensable in cells deficient in active p53 and inhibition of c-Abl reduced mitogen-induced c-myc expression. These data identify a novel function of cytoplasmic c-Abl in the signalling pathways regulating growth factor-induced c-myc expression and we propose the existence of a tyrosine kinase signalling cascade (PDGFR/c-Src/c-Abl) important for mitogenesis.

Imatinib mesylate (IM), a small molecule that is a selective inhibitor of the ABL, platelet derived growth factor receptor (PDGFR-R) and stem cell ligand receptor (c-kit) tyrosine kinases (TK). IM was also found to inhibit the TK activity of BCR/ABL fusion protein produced in chronic myelogenous leukemia, with marked clinical activity against the disease. Since both PDGF-R and c-kit both having a putative role in tumorigenesis, we investigated the efficacy and safety of the use of IM in patients with endocrine tumors unresponsive to conventional therapies that expressed c-kit and/or PDGF-R (within the framework of a comprehensive phase II multi-center study of IM in patients with solid tumors). IM was initiated at a dose of 400 mg/day, with possible dose escalation within 1 week to 600 mg/day and an option to raise the dose to 800 mg/day in the event of progression and in the absence of safety concerns for a period of up to 12 months. Between September 2002 and July 2003, 15 adult patients with disseminated endocrine tumors were recruited as follows: medullary thyroid carcinoma (MTC, n = 6); adrenocortical carcinoma (ACC, n = 4); malignant pheochromocytoma (pheo, n = 2); carcinoid (non-secreting, n = 2), neuroendocrine tumor (NET, n = 1). No objective responses were observed. MTC--disease progression in 4 patients, and treatment discontinuation in 2 patients due to adverse events; ACC--disease progression in 3 patients, and treatment discontinuation in 1 patient due to severe psychiatric adverse event; Pheo--disease progression in 2 patients; Carcinoid--stable disease in 1 patient (6.5 months), and disease progression in 1 patient; NET--disease progression in 1 patient. IM does not appear to be useful for treatment of malignant endocrine tumors, also causing significant toxicity in this patient population.

Platelet-derived growth factor (PDGF) stimulates many of the processes important in tissue repair, including proliferation of fibroblasts and synthesis of extracellular matrices. In this study we have demonstrated with in situ hybridization and immunocytochemistry the reversible expression of c-sis/PDGF-2 and PDGF receptor (PDGF-R) b mRNAs and their respective protein products in epithelial cells and fibroblasts following cutaneous injury in pigs. Epithelial cells in control, unwounded skin did not express c-sis and PDGF-R mRNAs, and fibroblasts expressed only PDGF-R mRNA. The expression levels in the injured site were correlated with the stage of tissue repair, being highest during the initial stages of the repair process and declining at the time of complete re-epithelialization and tissue remodeling. It is suggested that the controlled, reversible expression of a potent mitogen and its receptor induced by injury may function in an autocrine/paracrine manner on both epithelial cells and fibroblasts to bring about their sustained proliferation during the normal healing process. These studies provide a molecular basis for understanding the mechanisms contributing to normal tissue repair. We suggest the possibility that a defect in these mechanisms may be associated with defective wound healing. It is also conceivable that "chronic" injury may induce irreversible gene expression leading to pathologic, unregulated cell growth.

OBJECTIVE

Circulating progenitors and stem cells have been reported to contribute to angiogenesis and arterial repair after injury. In the present study, we investigated whether the arterial wall could host permanently residing progenitor cells under physiological context.

RESULTS

Using the Hoechst-based flow cytometry method, we identified and isolated progenitor cells termed side population (SP) cells at a prevalence of 6.0+/-0.8% in the tunica media of adult mice aortas. Arterial SP cells expressed the ATP-binding cassette transporter subfamily G member 2, frequently present on SP cell surface, and displayed a Sca-1+ c-kit(-/low) Lin- CD34(-/low) profile. They did not form myeloid or lymphoid hematopoietic colonies after plating in methylcellulose-based medium. Importantly, cultured SP cells were able to acquire the phenotype of endothelial cells (CD31, VE-cadherin, and von Willebrand factor expression) or of smooth muscle cells (alpha-smooth muscle actin, calponin, and smooth muscle myosin heavy chain expression), in presence of either vascular endothelial growth factor or transforming growth factor (TGF)-beta1/PDGF-BB, respectively. Moreover, they generated vascular-like branching structures, composed of both VE-cadherin+ cells and alpha-smooth muscle actin+ cells on Matrigel.

CONCLUSIONS

In this study, we provide the first evidence to our knowledge that in the adult mice, the normal arterial wall harbors SP cells with vascular progenitor properties.

Abnormal vascular smooth muscle cell (VSMC) proliferation is a key feature of atherosclerosis and restenosis; however, the mechanisms regulating growth remain unclear. Herein we show that inhibition of the aldehyde-metabolizing enzyme aldose reductase (AR) inhibits NF-kappa B activation during restenosis of balloon-injured rat carotid arteries as well as VSMC proliferation due to tumor necrosis factor alpha (TNF-alpha) stimulation. Inhibition of VSMC growth by AR inhibitors was not accompanied by increase in cell death or apoptosis. Inhibition of AR led to a decrease in the activity of the transcription factor NF-kappa B in culture and in the neointima of rat carotid arteries after balloon injury. Inhibition of AR in VSMC also prevented the activation of NF-kappa B by basic fibroblast growth factor (bFGF), angiotensin-II (Ang-II), and platelet-derived growth factor (PDGF-AB). The VSMC treated with AR inhibitors showed decreased nuclear translocation of NF-kappa B and diminished phosphorylation and proteolytic degradation of I kappa B-alpha. Under identical conditions, treatment with AR inhibitors also prevented the activation of protein kinase C (PKC) by TNF-alpha, bFGF, Ang-II, and PDGF-AB but not phorbol esters, indicating that AR inhibitors prevent PKC stimulation or the availability of its activator but not PKC itself. Treatment with antisense AR, which decreased the AR activity by >80%, attenuated PKC activation in TNF-alpha, bFGF, Ang-II, and PDGF-AB-stimulated VSMC and prevented TNF-alpha-induced proliferation. Collectively, these data suggest that inhibition of NF-kappa B may be a significant cause of the antimitogenic effects of AR inhibition and that this may be related to disruption of PKC-associated signaling in the AR-inhibited cells.