SH2 (src-homology 2) domains define a newly recognized binding motif that mediates the physical association of target phosphotyrosyl proteins with downstream effector enzymes. An example of such phosphoprotein-effector coupling is provided by the association of phosphatidylinositol 3-kinase (PI 3-kinase) with specific phosphorylation sites within the PDGF receptor, the c-Src/polyoma virus middle T antigen complex and the insulin receptor substrate IRS-1. Notably, phosphoprotein association with the SH2 domains of p85 also stimulates an increase in catalytic activity of the PI 3-kinase p110 subunit, which can be mimicked by phosphopeptides corresponding to targeted phosphoprotein phosphorylation sites. To investigate how phosphoprotein binding to the p85 SH2 domain stimulates p110 catalytic activation, we have examined the differential effects of phosphotyrosine and PDGF receptor-, IRS-1- and c-Src-derived phosphopeptides on the conformation of an isolated SH2 domain of PI 3-kinase. Although phosphotyrosine and both activating and non-activating phosphopeptides bind to the SH2 domain, activating phosphopeptides bind with higher affinity and induce a qualitatively distinct conformational change as monitored by CD and NMR spectroscopy. Amide proton exchange and protease protection assays further show that high affinity, specific phosphopeptide binding induces non-local dynamic SH2 domain stabilization. Based on these findings we propose that specific phosphoprotein binding to the p85 subunit induces a change in SH2 domain structure which is transmitted to the p110 subunit and regulates enzymatic activity by an allosteric mechanism.

Receptor tyrosine kinases (RTKs) have been implicated as therapeutic targets for the treatment of human diseases including cancers, inflammatory diseases, cardiovascular diseases including arterial restenosis, and fibrotic diseases of the lung, liver, and kidney. Three classes of 3-substituted indolin-2-ones containing propionic acid functionality attached to the pyrrole ring at the C-3 position of the core have been identified as catalytic inhibitors of the vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) RTKs. Some of the compounds were found to inhibit the tyrosine kinase activity associated with isolated vascular endothelial growth factor receptor 2 (VEGF-R2) [fetal liver tyrosine kinase 1 (Flk-1)/kinase insert domain-containing receptor (KDR)], fibroblast growth factor receptor (FGF-R), and platelet-derived growth factor receptor (PDGF-R) tyrosine kinase with IC(50) values at nanomolar level. Thus, compound 1 showed inhibition against VEGF-R2 (Flk-1/KDR) and FGF-R1 tyrosine kinase activity with IC(50) values of 20 and 30 nM, respectively, while compound 16f inhibited the PDGF-R tyrosine kinase activity with IC(50) value of 10 nM. Structural models and structure-activity relationship analysis of these compounds for the target receptors are discussed. The cellular activities of these compounds were profiled using cellular proliferation assays as measured by bromodeoxyuridine (BrdU) incorporation. Specific and potent inhibition of cell growth was observed for some of these compounds. These data provide evidence that these compounds can be used to inhibit the function of these target receptors.

BACKGROUND

Nanotechnology is a rapidly advancing industry with many new products already available to the public. Therefore, it is essential to gain an understanding of the possible health risks associated with exposure to nanomaterials and to identify biomarkers of exposure. In this study, we investigated the fibrogenic potential of SWCNT synthesized by chemical vapor deposition using cobalt (Co) and molybdenum (Mo) as catalysts. Following a single oropharyngeal aspiration of SWCNT in rats, we evaluated lung histopathology, cell proliferation, and growth factor mRNAs at 1 and 21 days post-exposure. Comparisons were made to vehicle alone (saline containing a biocompatible nonionic surfactant), inert carbon black (CB) nanoparticles, or vanadium pentoxide (V2O5) as a known inducer of fibrosis.

RESULTS

SWCNT or CB caused no overt inflammatory response at 1 or 21 days post-exposure as determined by histopathology and evaluation of cells (>95% macrophages) in bronchoalveolar lavage (BAL) fluid. However, SWCNT induced the formation of small, focal interstitial fibrotic lesions within the alveolar region of the lung at 21 days. A small fraction of alveolar macrophages harvested by BAL from the lungs of SWCNT-exposed rats at 21 days were bridged by unique intercellular carbon structures that extended into the cytoplasm of each macrophage. These "carbon bridge" structures between macrophages were also observed in situ in the lungs of SWCNT-exposed rats. No carbon bridges were observed in CB-exposed rats. SWCNT caused cell proliferation only at sites of fibrotic lesion formation as measured by bromodeoxyuridine uptake into alveolar cells. SWCNT increased platelet-derived growth factor (PDGF)-A, PDGF-B, and PDGF-C mRNA levels significantly at 1 day as measured by Taqman quantitative real-time RT-PCR. At 21 days, SWCNT did not increase any mRNAs evaluated, while V2O5 significantly increased mRNAs encoding PDGF-A, -B, and -C chains, PDGF-R alpha, osteopontin (OPN), connective tissue growth factor (CTGF), and transforming growth factor (TGF)-beta1.

CONCLUSIONS

Our findings indicate that SWCNT do not cause lung inflammation and yet induce the formation of small, focal interstitial fibrotic lesions in the alveolar region of the lungs of rats. Of greatest interest was the discovery of unique intercellular carbon structures composed of SWCNT that bridged lung macrophages. These "carbon bridges" offer a novel and easily identifiable biomarker of exposure.

Upon liver injury, hepatic stellate cells (HSC) show increased proliferation, motility, and extracellular matrix (ECM) production. The extracellular signal-regulated kinases (ERK) control different functions in a cell-specific manner. In this study, we evaluated the role of ERK activation in cultured HSC stimulated with platelet-derived growth factor (PDGF) and after induction of liver injury in vivo. HSC were isolated from normal human liver tissue, cultured on plastic, and used in their myofibroblast-like phenotype. In in vivo experiments, HSC were isolated from normal rats or at different time points after a single intragastric administration of CCl(4). Nontoxic concentrations of PD98059, a specific inhibitor of ERK activation, reduced PDGF-induced activation of ERK in a dose-dependent fashion. Suppression of ERK activation was associated with complete inhibition of HSC proliferation and with a 57% reduction in chemotaxis. In the presence of the ERK inhibitor, binding of the AP-1 complex and of STAT1 to the related regulatory elements was inhibited. The inhibition of the DNA binding activity of STAT1 was mediated by a reduction in PDGF-induced tyrosine phosphorylation. Expression of c-fos in response to PDGF was also reduced, but not suppressed, by treatment with PD98059. In HSC isolated from CCl(4)-treated rats, ERK activity increased as early as 6 hours following liver damage, and declined thereafter. The results of this study indicate that ERK activation regulates proliferation and chemotaxis of HSC, and modulates nuclear signaling. Acute liver damage in vivo leads to activation of ERK in HSC.

Small cell lung cancer (SCLC) is an aggressive cancer characterized by several autocrine growth mechanisms including stem cell factor and its receptor c-Kit. In order to arrive at potentially new and novel therapy for SCLC, we have investigated the effects of the tyrosine kinase inhibitor, STI 571, on SCLC cell lines. It has been previously reported that STI 571 does not only inhibit cellular Abl tyrosine kinase activity but also the PDGF receptor and c-Kit tyrosine kinases at similar concentrations (approximately 0.1 microM). There is no expression of the PDGF-receptor, and the Abl kinase is not activated by SCLC, but over 70% of SCLC contain the c-Kit receptor. Utilizing this preliminary data, we have determined that three (NCI-H69, NCI-H146 and NCI-H209) of five (including NCI-H82 and NCI-H249) SCLC cell lines had detectable c-Kit receptors and were inhibited in growth and viability at concentrations 1 - 5 microM of STI 571 after 48 h of treatment. The SCLC cell lines, NCI-H69, NCI-H146 and NCI-H209, showed a dose-response (tested between 0.1 - 10 microM) inhibition of tyrosine phosphorylation of c-Kit as well as in vitro kinase activity (at 5 microM) of c-Kit in response to STI 571. STI 571 inhibited cell motility, as assessed by time-lapsed video microscopy, within 6 h of STI 571 treatment (5 microM). STI 571 also decreased intracellular levels of reactive oxygen species (ROS) by at least 60%, at a concentration (5 microM) that also inhibited cell growth. Cell cycle analysis of STI 571 responsive cells showed that cells were generally slowed in G2/M phase, but there was no arrest at G1/S. A downstream phosphorylation target of c-Kit, Akt, was not phosphorylated in response to stem cell factor in the presence of STI 571. These data imply that STI 571 inhibits growth of SCLC cells through a mechanism that involves inactivation of the tyrosine kinase c-Kit. The effectiveness of STI 571 in this study suggests this drug may be useful in a clinical trial, for patients with SCLC. Oncogene (2000) 19, 3521 - 3528

The tyrosine kinase inhibitor STI571 inhibits BCR/ABL and induces hematologic remission in most patients with chronic myeloid leukemia. In addition to BCR/ABL, STI571 also inhibits v-Abl, TEL/ABL, the native platelet-derived growth factor (PDGF)beta receptor, and c-KIT, but it does not inhibit SRC family kinases, c-FMS, FLT3, the epidermal growth factor receptor, or multiple other tyrosine kinases. ARG is a widely expressed tyrosine kinase that shares substantial sequence identity with c-ABL in the kinase domain and cooperates with ABL to regulate neurulation in the developing mouse embryo. As described here, ARG has recently been implicated in the pathogenesis of leukemia as a fusion partner of TEL. A TEL/ARG fusion was constructed to determine whether ARG can be inhibited by STI571. When expressed in the factor-dependent murine hematopoietic cell line Ba/F3, the TEL/ARG protein was heavily phosphorylated on tyrosine, increased tyrosine phosphorylation of multiple cellular proteins, and induced factor-independent proliferation. The effects of STI571 on Ba/F3 cells transformed with BCR/ABL, TEL/ABL, TEL/PDGFbetaR, or TEL/ARG were then compared. STI571 inhibited tyrosine phosphorylation and cell growth of Ba/F3 cells expressing BCR/ABL, TEL/ABL, TEL/PDGFbetaR, and TEL/ARG with an IC(50) of approximately 0.5 microM in each case, but it had no effect on untransformed Ba/F3 cells growing in IL-3 or on Ba/F3 cells transformed by TEL/JAK2. Culture of TEL/ARG-transfected Ba/F3 cells with IL-3 completely prevented STI571-induced apoptosis in these cells, similar to what has been observed with BCR/ABL- or TEL/ABL-transformed cells. These results indicate that ARG is a target of the small molecule, tyrosine kinase inhibitor STI571.

OBJECTIVE

To compare the mRNA expression of growth factor receptors in cultured human trabecular meshwork (HTM) cells with ex vivo HTM tissues and to determine whether HTM cells generate a physiologic response after exposure to exogenous growth factors.

METHODS

The reverse transcription-polymerase chain reaction (RT-PCR) method was used to detect the expression of various growth factor receptor mRNAs using early passaged, cultured HTM cells from donors of several ages. RT-PCR on ex vivo HTM tissues from healthy donors and donors with glaucoma were also used to compare and contrast mRNA expression with cell culture results. After the exogenous administration of growth factors, cell proliferation and extracellular acidification rate studies were used to measure the functional responses of HTM cells to growth factors.

RESULTS

Amplification products of the expected size for 15 growth factor receptors were detected in cultured HTM cells and in ex vivo HTM tissues. The administration of exogenous growth factors showed that (a) hepatocyte growth factor (HGF), epidermal growth factor (EGF), insulinlike growth factor (IGF)-1, tumor necrosis factor (TNF) alpha, platelet-derived growth factor (PDGF)-AA, PDGF-BB, PDGF-AB, and basic fibroblast growth factor (FGF-2) stimulated cell proliferation, whereas FGF-1 (acidic), transforming growth factor (TGF) alpha, interleukin (IL)-1alpha, nerve growth factor (NGF), and FGF-7 (keratinocyte growth factor [KGF]) had no significant influence on cell proliferation; (b) TGF-beta isoforms significantly inhibited EGF-stimulated trabecular meshwork cell proliferation; and (c) FGF-1 (acidic), TGF-alpha, EGF, IL-1alpha, IL-1beta, HGF, TNF-alpha, PDGF-AA, and IGF-1 significantly stimulated extracellular acidification, whereas FGF-2 (basic), FGF-7 (KGF), TGF-beta1-beta3 and NGF had no significant influence on extracellular acidification.

CONCLUSIONS

These studies show that mRNA for numerous growth factor receptors can be detected in cultured HTM cells and in ex vivo HTM tissues. They also show that many of the receptors are functional, because exogenous growth factor administration elicits a physiologic response. In vivo, these receptors may be activated by growth factors present within the aqueous humor (aquecrine/paracrine) or by growth factors synthesized and released locally by trabecular meshwork cells themselves (autocrine). Specific growth factors acting through high-affinity receptors may be involved in maintaining the normal microenvironment of the HTM and also may be involved in the pathogenesis of primary open-angle glaucoma.

The Slits are secreted proteins with roles in axonal guidance and leukocyte migration. On binding to Robo receptors, Slit2 repels developing axons and inhibits leukocyte chemotaxis. Slit2 is cleaved into Slit2-N, a protein tightly binding to cell membranes, and Slit2-C, a diffusible fragment. In the present study, we characterized the functional role of Slit2-N in vascular smooth muscle cells (VSMCs) and the cell association properties of 2 truncated versions of Slit2-N. Here, we document for the first time that Slit2-N is a chemorepellent of VSMCs. Intact blood vessels expressed Slit2 and Robo receptors as demonstrated by immunohistochemistry and quantitative real time PCR. Recombinant Slit2-N prevented the platelet-derived growth factor (PDGF)-stimulated migration of VSMCs. Slit2-N also abrogated PDGF-mediated activation of small guanosine triphosphatase (GTPase) Rac1, a member of the Rho GTPase superfamily of proteins involved in regulating the actin cytoskeleton. Furthermore, Slit2-N inhibited the PDGF-induced formation of lamellipodia, a crucial cytoskeletal reorganization event for cell motility. Slit2-N had no effect on the PDGF-mediated increase in DNA synthesis determined by [3H]thymidine uptake, suggesting that VSMC growth is unaffected by Slit2. Analysis of 2 engineered Slit2-N fragments (Slit2-N/1118 and Slit2-N/1121) indicated that 3 amino acids upstream of the putative cleavage site (Arg1121, Thr1122) are involved in the association of Slit2-N to the cell membrane. Our data assign a novel functional role to Slit2 in vascular function and show that cell guidance mechanisms that operate in the developing central nervous system are conserved in VSMCs.

We have investigated the regulation of mRNA levels for thrombospondin, a platelet-derived growth factor (PDGF)-regulated secreted glycoprotein, in cultures of rat vascular smooth muscle cells (SMC). A thrombospondin cDNA hybridizes to a single 5.5-kilobase SMC message which is greatly induced by serum or PDGF. When quiescent SMC are treated with PDGF, thrombospondin levels are induced rapidly (within 15 min) and in a dose-dependent manner. The induction of thrombospondin message levels parallels PDGF-mediated mitogenesis. PDGF-mediated increases in thrombospondin mRNA can be blocked in the presence of actinomycin D, suggesting that PDGF regulates the thrombospondin message at the level of gene transcription. In the presence of cycloheximide, PDGF "super-induces" the thrombospondin message. The data establish that mRNA levels for thrombospondin are regulated by PDGF in a manner similar to that of c-myc, c-fos, and other growth-regulatory gene products and extend our previous findings that thrombospondin secretion by SMC is dependent on exposure to PDGF. These observations are strongly suggestive of an important role for thrombospondin in the growth response of mesenchymal cells.

Src homology 2 (SH2) domains are found in a variety of signaling proteins and bind phosphotyrosine-containing peptide sequences. To explore the binding properties of the SH2 domain of the Src protein kinase, we used immobilized phosphopeptides to bind purified glutathione S-transferase-Src SH2 fusion proteins. With this assay, as well as a free-peptide competition assay, we have estimated the affinities of the Src SH2 domain for various phosphopeptides relative to a Src SH2-phosphopeptide interaction whose Kd has been determined previously (YEEI-P; Kd = 4 nM). Two Src-derived phosphopeptides, one containing the regulatory C-terminal Tyr-527 and another containing the autophosphorylation site Tyr-416, bind the Src SH2 domain in a specific though low-affinity manner (with about 10(4)-lower affinity than the YEEI-P peptide). A platelet-derived growth factor receptor (PDGF-R) phosphopeptide containing Tyr-857 does not bind appreciably to the Src SH2 domain, suggesting it is not the PDGF-R binding site for Src as previously reported. However, another PDGF-R-derived phosphopeptide containing Tyr-751 does bind the Src SH2 domain (with an affinity approximately 2 orders of magnitude lower than that of YEEI-P). All of the phosphopeptides which bind to the Src SH2 domain contain a glutamic acid at position -3 or -4 with respect to phosphotyrosine; changing this residue to alanine greatly diminishes binding. We have also tested Src SH2 mutants for their binding properties and have interpreted our results in light of the recent crystal structure solution for the Src SH2 domain. Mutations in various conserved and nonconserved residues (R155A, R155K, N198E, H201R, and H201L) cause slight reductions in binding, while two mutations cause severe reductions. The W148E mutant domain, which alters the invariant tryptophan that marks the N-terminal border of the SH2 domain, binds poorly to phosphopeptides. Inclusion of the SH3 domain in the fusion protein partially restores the binding by the W148E mutant. A change in the invariant arginine that coordinates twice with phosphotyrosine in the peptide (R175L) results in a nearly complete loss of binding. The R175L mutant does display high affinity for the PDGF-R peptide containing Tyr-751, via an interaction that is at least partly phosphotyrosine independent. We have used this interaction to show that the R175L mutation also disrupts the intramolecular interaction between the Src SH2 domain and the phosphorylated C terminus within the context of the entire Src protein; thus, the binding properties observed for mutant domains in an in vitro assay appear to mimic those that occur in vivo.

1. Extracellular adenosine triphosphate (ATP) is mitogenic for vascular smooth muscle cells (VSMC) and stimulates several events that are important for cell proliferation: DNA synthesis, protein synthesis, increase of cell number, immediate early genes, cell-cycle progression, and tyrosine phosphorylation. 2. Receptor characterization indicates mitogenic effects of both P2U and P2Y receptors. The P2X receptor is lost in cultured VSMC and is not involved. Several related biological substances such as UTP, ITP, GTP, AP4A, ADP, and UDP are also mitogenic. 3. Signal transduction is mediated via Gq-proteins, phospholipase C beta, phospholipase D, diacyl glycerol, protein kinase C alpha, delta, Raf-1, MEK, and MAPK. 4. ATP acts synergistically with polypeptide growth factors (PDGF, bFGF, IGF-1, EGF, insulin) and growth factors acting via G-protein-coupled receptors (noradrenaline, neuropeptide Y, 5-hydroxytryptamine, angiotensin II, endothelin-1). 5. The mitogenic effects have been demonstrated in rat, porcine, and bovine VSMC and cells from human coronary arteries, aorta, and subcutaneous arteries and veins. 6. The trophic effects on VSMC and the abundant sources for extracellular ATP in the vessel wall make a pathophysiological role probable in the development of atherosclerosis, neointima-formation after angioplasty, and possibly hypertension.

Cardiac fibroblasts appear to be important in producing and maintaining the extracellular matrix (ECM) of the heart. The abnormal proliferation of cardiac fibroblasts and deposition of the ECM protein, collagen, associated with hypertension and myocardial infarction, may adversely affect the performance of the heart. Several groups of factors affect collagen gene expression and/or growth of cardiac fibroblasts. Angiotensin II, aldosterone and endothelins play a central role in the remodeling of the ECM in hypertension, and decrease collagenase activity and/or increase collagen synthesis in cultured cells. Regulatory peptides that are generally elevated at sites of injury, such as TGF-beta 1 and PDGF, increase collagen synthesis and/or stimulate mitogenesis. Mechanical stretch enhances collagen expression and cell proliferation, responses which could in part be due to integrin activation. Cytokines may stimulate or inhibit cell growth, the latter through prostaglandin formation. Angiotensin II is a principal determinant in vivo of cardiac fibroplasia and synthesis of the ECM proteins, collagen and fibronectin. Cardiac fibroblasts possess G-protein-coupled AT1 receptors for angiotensin II that couple to activation of multiple signalling pathways, including: phospholipase C-beta, with the subsequent release of Ca2+ from intracellular stores and activation of protein kinase C, mitogen-activated protein kinases, tyrosine kinases, phospholipase D, phosphatidic acid formation, and the STAT family of transcription factors. Cardiac fibroblasts respond to angiotensin II with hyperplastic/hypertrophic growth, and increased expression of collagen, fibronectin, and integrins. The mechanisms by which the AT1 receptor activates multiple signalling pathways are not known, although the receptor might interact at some level with both integrins and cytokine receptors. Different signalling pathways of the AT1 receptor may subserve different cellular responses, such as mitogenesis, ECM synthesis, or an inflammatory/stress response. Crosstalk among the signalling pathways of the AT1 receptor, and those of G-protein, cytokine, and growth-factor receptors, may determine the ultimate response of the cell.

OBJECTIVE

Activation of the platelet-derived growth factor (PDGF) and c-kit receptors has been proposed as important in mediating the growth of AIDS-related Kaposi's sarcoma (KS). We investigated the response of KS to the PDGF receptor (PDGFR)/c-kit inhibitor, imatinib mesylate, and investigated the effect of this therapy on critical signal transduction intermediates.

METHODS

Ten male patients with AIDS-related cutaneous KS, which progressed despite chemotherapy and/or highly active antiretroviral therapy, received imatinib mesylate administered orally, 300 mg twice daily. Clinical response was determined by serial tumor measurements. To determine biologic and histologic response, skin lesion biopsies were obtained at baseline and following 4 weeks of therapy.

RESULTS

Five of 10 participants had a partial response by tumor measurements. Biopsies after 4 weeks of therapy demonstrated histologic regression in four of six patients. Four patients' tumor biopsies were assessable for immunohistochemistry end points pre- and post-therapy. These demonstrated inhibition of PDGFR and its downstream effector, extracellular receptor kinase, which is a member of the mitogen-activated protein kinase family. The most common adverse event was diarrhea, which led to dose reduction in six patients.

CONCLUSIONS

Imatinib mesylate administered orally twice daily for AIDS-related KS results in clinical and histologic regression of cutaneous KS lesions within 4 weeks. These promising results demonstrate that inhibition of the c-kit and/or PDGF receptors may represent an effective strategy for treating KS.

The treatment of advanced renal cell carcinoma (RCC) has undergone a major change with the development of potent angiogenesis inhibitors and targeted agents. Several multitargeted tyrosine kinase inhibitors, sorafenib and sunitinib, have already been approved for the treatment of advanced RCC. Temsirolimus (CCI-779), a mammalian target of rapamycin inhibitor, has shown a survival advantage over IFN in advanced, poor-prognosis RCC patients. Bevacizumab, an antibody targeting vascular endothelial growth factor (VEGF) A, has also shown promising clinical activity. Benefits attributable to these agents have been recognized by high objective response rates (sunitinib), significant increases in progression-free survival (sunitinib, sorafenib and bevacizumab), or improved overall survival (temsirolimus). These agents mediate much of their effect through inhibition of the hypoxia-inducible factor (HIF)-VEGF-VEGF receptor axis. Their inhibitory activity for the signaling of platelet-derived growth factor (PDGF) receptor beta or kinases like c-Raf may contribute to the antitumor effects of the multitargeted kinase inhibitors. Nevertheless, all four single agents rarely, if ever, induce complete responses and, at present, all patients develop resistance and, ultimately, progress during therapy. A critical need exists to develop strategies that may increase the degree of the antitumor effects with the hope of inducing more complete responses impeding the onset of or elimination of refractory disease. Combinations of these and other targeted agents may overcome the resistance that develops with single-agent therapy and could be incorporated either as part of initial therapy or later when disease resistance develops. Approaches aimed at combining these agents can be based on the genetics and biology of clear cell RCC. von Hippel-Lindau loss leads to an increase in cellular levels of HIF (HIF-1alpha or HIF-2alpha) leading to increased expression of a number of hypoxia-regulated genes critical to cancer progression. Combinations of targeted agents may block several of these mediators (VEGF, epidermal growth factor receptor, and PDGF), so-called horizontal blockade. Blockade could also take place at two levels of the pathways (vertical blockade), either at HIF and VEGF or at VEGF and VEGF receptor signaling. Many of the above strategies are ongoing and will require careful phase 1 determination of toxicity and even more rigorous phase 2 analysis before moving onto phase 3 trials.

The c-Abl nonreceptor tyrosine kinase is activated by growth factor signals such as the platelet-derived growth factor (PDGF) and functions downstream of the PDGF-beta receptor (PDGFR) to mediate biological processes such as membrane ruffling, mitogenesis, and chemotaxis. Here, we show that the related kinase Arg is activated downstream of PDGFRs in a manner dependent on Src family kinases and phospholipase C gamma1 (PLC-gamma1)-mediated phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis, as we showed previously for c-Abl. PIP2, a highly abundant phosphoinositide known to regulate cytoskeletal and membrane proteins, inhibits the tyrosine kinase activities of both Arg and c-Abl in vitro and in cells. We now demonstrate that c-Abl and Arg form inducible complexes with and are phosphorylated by the PDGFR tyrosine kinase in vitro and in vivo. Moreover, c-Abl and Arg, in turn, phosphorylate the PDGFR. We show that c-Abl and Arg exhibit nonredundant functions downstream of the activated PDGFR. Reintroduction of c-Abl into Arg-Abl double-null fibroblasts rescues the ability of PLC-gamma1 to increase PDGF-mediated chemotaxis, while reexpression of Arg fails to rescue the chemotaxis defect. These data show that, although both kinases are activated and form complexes with proteins in the PDGFR signaling pathway, only c-Abl functions downstream of PLC-gamma1 to mediate chemotaxis.

Cholangiocarcinoma (CCA) is characterized by an abundant stromal reaction. Cancer-associated fibroblasts (CAFs) are pivotal in tumor growth and invasiveness and represent a potential therapeutic target. To understand the mechanisms leading to CAF recruitment in CCA, we studied (1) expression of epithelial-mesenchymal transition (EMT) in surgical CCA specimens and CCA cells, (2) lineage tracking of an enhanced green fluorescent protein (EGFP)-expressing human male CCA cell line (EGI-1) after xenotransplantation into severe-combined-immunodeficient mice, (3) expression of platelet-derived growth factors (PDGFs) and their receptors in vivo and in vitro, (4) secretion of PDGFs by CCA cells, (5) the role of PDGF-D in fibroblast recruitment in vitro, and (6) downstream effectors of PDGF-D signaling. CCA cells expressed several EMT biomarkers, but not alpha smooth muscle actin (α-SMA). Xenotransplanted CCA masses were surrounded and infiltrated by α-SMA-expressing CAFs, which were negative for EGFP and the human Y-probe, but positive for the murine Y-probe. CCA cells were strongly immunoreactive for PDGF-A and -D, whereas CAFs expressed PDGF receptor (PDGFR)β. PDGF-D, a PDGFRβ agonist, was exclusively secreted by cultured CCA cells. Fibroblast migration was potently induced by PDGF-D and CCA conditioned medium and was significantly inhibited by PDGFRβ blockade with Imatinib and by silencing PDGF-D expression in CCA cells. In fibroblasts, PDGF-D activated the Raccc-Jun N-terminal kinase (JNK). Selective inhibition of Rho GTPases (particularly Racced PDGF-D-induced fibroblast migration.

CONCLUSIONS

CCA cells express several mesenchymal markers, but do not transdifferentiate into CAFs. Instead, CCA cells recruit CAFs by secreting PDGF-D, which stimulates fibroblast migration through PDGFRβ and Rho GTPase and JNK activation. Targeting tumor or stroma interactions with inhibitors of the PDGF-D pathway may offer a novel therapeutic approach.

Many extracellular and intrinsic factors regulate oligodendrocyte development, but their signaling pathways remain poorly understood. Although the p38 mitogen-activated protein kinase (MAPK)-dependent pathway is implicated in oligodendrocyte progenitor cell (OPC) lineage progression, its molecular targets involved in myelinogenesis are mostly unidentified. We have analyzed mechanisms by which p38MAPK regulates oligodendrocyte development and demonstrate that p38MAPK inhibition prevents OPC lineage progression and inhibits MBP (myelin basic protein) promoter activity and Sox10 function. In white-matter tissue, differential levels of MAPK phosphorylation are observed in oligodendrocyte lineage cells. Phosphorylated p38MAPK was found in CC1- and CNP-expressing differentiated oligodendrocytes of the adult brain and was temporally associated with a decline in the levels of phosphorylated extracellular signal-regulated kinase (ERK) in cells of this lineage. PDGF stimulates the phosphorylation of ERK, p38MAPK, and c-Jun N-terminal kinase (JNK), and p38MAPK inhibition was associated with increased ERK, JNK, and c-Jun phosphorylation. In the presence of PDGF, simultaneous inhibition of p38MAPK and either MAPK kinase (MEK) or JNK significantly alleviates the repression of myelin gene expression and lineage progression induced by p38MAPK inhibition alone. Dominant-negative c-Jun reverses the inhibition of myelin promoter activity by active MEK1 or dominant-negative p38MAPKalpha mutants, and phosphorylated c-Jun was detected at the MBP promoter after p38MAPK inhibition, indicating c-Jun as a negative mediator of p38MAPK action. Our findings indicate that p38MAPK activity in the brain supports myelin gene expression through distinct mechanisms via positive and negative regulatory targets. We show that oligodendrocyte differentiation involves p38-mediated Sox10 regulation and cross talk with parallel ERK and JNK pathways to repress c-Jun activity.

The platelet-derived growth factor (PDGF)-A promoter is regulated by a number of GC-rich regulatory elements that possess non-B-form DNA structures. Screening of a HeLa cDNA expression library with the C-rich strand of a PDGF-A silencer sequence (5'-S1 nuclease-hypersensitive site (SHS)) yielded three cDNA clones encoding NM23-H1, a protein implicated as a suppressor of metastasis in melanoma and breast carcinoma. Recombinant human NM23-H1 cleaved within the 3'-portions of both 5'-SHS strands in either single-stranded or duplex forms. In contrast, NM23-H2, known as a transcriptional activator with a DNA cleavage function, cleaved within the 5'-portions of both strands, revealing that NM23-H1 and NM23-H2 cleave at distinct sites of the 5'-SHS and by different mechanisms. NM23-H1 and NM23-H2 also cleaved within the PDGF-A basal promoter region, again exhibiting preferences for cleavage within the 5'- and 3'-portions of the element, respectively. Transient transfection analyses in HepG2 cells revealed that both NM23-H1 and -H2 repressed transcriptional activity driven by the PDGF-A basal promoter (-82 to +8). Activity of the negative regulatory region (-1853 to -883), which contains the 5'-SHS, was also inhibited modestly by NM23-H1 and NM23-H2. These studies demonstrate for the first time that NM23-H1 interacts both structurally and functionally with DNA. They also indicate a role for NM23 proteins in repressing transcription of a growth factor oncogene, providing a possible molecular mechanism to explain their metastasis-suppressing effects.

Platelet-derived growth factor (PDGF) protein family members are potent mitogens and chemoattractants for mesenchymal cells. The classic PDGF ligands A and B are single-domain protein chains which are secreted as active dimers capable of activating their cognate PDGF receptors (PDGFRs). In contrast to PDGFs A and B, PDGF D contains an N-terminal complement subcomponent CCCUB) domain and a C-terminal PDGF domain. PDGF D must undergo extracellular proteolytic processing, separating the CUB domain from the PDGF domain, before the PDGF domain can stimulate beta-PDGFR-mediated cell signal transduction. Here, we report that prostate carcinoma cells LNCaP and PCPDGF D into its active form under serum-independent conditions and that this autoactivation is inhibited by PAI-1, a urokinase plasminogen activator (uPA)/tissue plasminogen activator (tPA) inhibitor. Interestingly, uPA, but not the closely related protease tPA, is capable of processing recombinant latent PDGF DD into the active form. We identify the uPA cleavage site between the CUB and PDGF domains of the full-length PDGF D by mutational analysis and show that PDGF D and uPA colocalize in human prostate carcinoma. This evidence provides a direct link between uPA- and PDGF D-mediated cell signaling, which may contribute to the progression of prostate cancer.

Recent developments in the molecular biology of the insulin-like growth factor I (IGF-I) receptor have clarified its role in cellular growth and transformation. Although cells homozygous for a targeted disruption of the IGF-I receptor genes can grow in serum-supplemented medium, the IGF-I receptor is required for optimal growth, and is required equally in all phases of the cell cycle. The receptor plays an even more stringent role in cellular transformation and tumorigenicity, which seem to be dependent on its normal expression in several cell types. The expression of both the IGF-I receptor and its ligands is regulated by other growth factors (especially PDGF and EGF), by oncogenes (like SV40 T antigen and c-myb) and by tumour suppressor genes (like WT1 and RB). The picture emerging from these studies is that several transforming agents may exert their growth promoting effects through the direct or indirect activation of the IGF autocrine loop.

Preincubation of Swiss 3T3 cells or human fibroblasts with purified platelet-derived growth factor (PDGF) at 4 degrees C or 37 degrees C rapidly inhibits subsequent binding of 125I-epidermal growth factor (125I-EGF). The effect does not result from competition by PDGF for binding to the EGF receptor since (a) very low concentrations of PDGF are effective, (b) cells with EGF receptors but no PDGF receptors are not affected, and (c) the inhibition persists even if the bound PDGF is eluted before incubating the cells with 125I-EGF. PDGF does not affect 125I-insulin binding nor does EGF affect 125I-PDGF binding under these conditions. Endothelial cell-derived growth factor also competes for binding to PDGF receptors and inhibits 125I-EGF binding. The inhibition demonstrated by PDGF seems to result from an increase in the Kd for 125I-EGF binding with no change in the number of EGF receptors.

Hepatocyte growth factor/scatter factor (HGF) and the angiogenesis factors platelet-derived growth factors (PDGF), vascular endothelial growth factor (VEGF), and interleukin-8 (IL-8) are found in elevated concentrations in serum or tumor tissue of patients with head and neck squamous cell carcinomas (HNSCC), suggesting these factors may be coregulated. A cDNA microarray analysis for HGF-inducible genes revealed that HGF also modulates PDGFA expression, a gene recently shown to be inducible by the transcription factor, early growth response-1 (Egr-1). In the present study, we investigated the potential role of HGF-induced Egr-1 in expression of PDGF, VEGF, and IL-8. HGF induced expression of all three factors and Egr-1 expression and DNA-binding activity. The analysis of promoter sequences showed putative Egr-1 binding sites in the PDGFA or VEGF but not in the IL-8 promoter, and HGF-induced Egr-1-binding activity was confirmed by chromatin immunoprecipitation (ChIP) assay. The maximal basal and HGF-induced promoter activity for the PDGFA gene existed within -630 bp of the promoter region, and overexpression of Egr-1 significantly increased such activity. Consistent with this, expression of PDGFA and VEGF but not IL-8 showed corresponding differences with Egr-1 expression in HNSCC tumor specimens and were strongly suppressed by transfection of Egr-1-antisense or small interference RNA (siRNA) oligonucleotides. HGF-induced expression of Egr-1, PDGFA, and VEGF was suppressed by pharmacologic and siRNA inhibitors of mitogen-activated protein kinase kinase 1/2 (MEK1/2) and protein kinase C (PKC) pathways. We conclude that the HGF-induced activation of transcription factor Egr-1 by MEK1/2- and PKC-dependent mechanisms differentially contributes to expression of PDGF and VEGF, which are important angiogenesis factors and targets for HNSCC therapy.

Malignant astrocytoma is the most common primary human brain tumor. Most astrocytomas express a combination of platelet-derived growth factor (PDGF) and PDGF receptor which could close an autocrine loop. It is not known whether these autocrine loops contribute to the transformed phenotype of astrocytoma cells or are incidental to that phenotype. Here we show that dominant-negative mutants of the PDGF ligand break the autocrine loop and revert the phenotype of BALB/c 3T3 cells transformed by the PDGF-A or PDGF-B (c-sis) gene. Then, we show that these mutants are selective in that they do not alter the phenotype of 3T3 cells transformed by an activated Ha-ras or v-src gene or by simian virus 40. Finally, we show that these mutants revert the transformed phenotype of two independent human astrocytoma cell lines. They have no effect on the growth of human medulloblastoma, bladder carcinoma, or colon carcinoma cell lines. These observations are consistent with the view that PDGF autocrine loops contribute to the transformed phenotype of at least some human astrocytomas.

It is shown here that the myogenic cell line L6J1, primary skeletal myoblasts, and primary adult arterial smooth muscle cells express the gene for the A chain but not the gene for the B chain (c-sis) of platelet-derived growth factor (PDGF). It is further demonstrated that conditioned media from L6J1 cultures contain material that (i) competes with 125I-labeled PDGF for binding to human fibroblasts, (ii) is specifically precipitated by antibodies against PDGF, and (iii) has a relative molecular mass comparable to that of PDGF and, after reduction, its constituent subunit chains. The secretion of PDGF-receptor-competing activity was at a maximum in exponentially growing cultures but remained at a high level also after the cells had become confluent, stopped dividing, and fused to form multinucleate myotubes. Similarly, it was previously demonstrated that adult rat arterial smooth muscle cells in primary culture produce a mitogenic protein with immunological and structural properties similar to PDGF. In accordance with these findings, it was recently shown that secretion of PDGF-like mitogens by a number of human tumor cell lines correlates with expression of the gene for the A chain rather than the B chain of PDGF. The results suggest that production of homodimers of PDGF A chains may stimulate proliferation of skeletal myoblasts and arterial smooth muscle cells in an autocrine or paracrine manner. This could fulfill important functions during myogenesis in the embryo as well as in tissue repair and atherogenesis in the adult.