The mechanisms through which the small GTPases Rac1 and Cdc42 regulate the formation of membrane ruffles, lamellipodia, and filopodia are currently unknown. The p21-activated kinases (PAKs) are direct targets of active Rac and Cdc42 which can induce the assembly of polarized cytoskeletal structures when expressed in fibroblasts, suggesting that they may play a role in mediating the effects of these GTPases on cytoskeletal dynamics. We have examined the subcellular localization of endogenous PAK1 in fibroblast cell lines using specific PAK1 antibodies. PAK1 is detected in submembranous vesicles in both unstimulated and stimulated fibroblasts that colocalize with a marker for fluid-phase uptake. In cells stimulated with PDGF, in v-Src-transformed fibroblasts, and in wounded cells, PAK1 redistributed into dorsal and membrane ruffles and into the edges of lamellipodia, where it colocalizes with polymerized actin. PAK1 was also colocalized with F-actin in membrane ruffles extended as a response to constitutive activation of Rac1. PAK1 appears to precede F-actin in translocating to cytoskeletal structures formed at the cell periphery. The association of PAK1 with the actin cytoskeleton is prevented by the actin filament-disrupting agent cytochalasin D and by the phosphatidylinositol 3-kinase inhibitor wortmannin. Co-immunoprecipitation experiments demonstrate an in vivo interaction of PAK1 with filamentous (F)-actin in stimulated cells. Microinjection of a constitutively active PAK1 mutant into Rat-1 fibroblasts overexpressing the insulin receptor (HIRcB cells) induced the formation of F-actin- and PAK1-containing structures reminiscent of dorsal ruffles. These data indicate a close correlation between the subcellular distribution of endogenous PAK1 and the formation of Rac/Cdc42-dependent cytoskeletal structures and support an active role for PAK1 in regulating cortical actin rearrangements.

Blood-brain barrier (BBB) disruption, mediated through matrix metalloproteinases (MMPs) and other mechanisms, is a critical event during ischemic stroke. Tissue plasminogen activator (tPA) is the only FDA-approved thrombolytic therapy for acute ischemic stroke, but the efficacy and safety of its therapeutic application are limited by narrow treatment time windows and side effects. Thus, there is a pressing need to develop combinational therapy that could offset tPA side effects and improve efficacy in clinical practice. Recent experimental studies indicate that tPA has previously unidentified functions in the brain beyond its well-established thrombolytic activity, which might contribute to tPA-related side effects through MMPs (mainly MMP-9) and several signaling pathways involved in LDL receptor-related protein (LRP), activated protein C (APC) and protease-activated receptor 1 (PAR-1), platelet-derived growth factor C (PDGF-C), and N-methyl-d-aspartate (NMDA) receptor. Therapeutic targeting of MMPs and/or tPA-related signaling pathways might offer promising new approaches to combination therapies for ischemic stroke. This review provides an overview of the relationship between structural components and function of the BBB/neurovascular unit with respect to ischemic stroke. We discuss how MMPs and tPA contribute to BBB disruption during ischemic stroke and highlight recent findings of molecular signaling pathways involved in neurotoxicity of tPA therapy.

Prostate cancer (PCa) has a predilection to metastasize to bone. Before metastasis can occur there is transition of the sessile epithelial cancer cells to become motile and invasive mesenchymal phenotypes by an important albeit transient process called Epithelial-to-Mesenchymal Transition (EMT). The cascade of molecular events triggered by this process is clinically relevant as they are associated with cancer stem-like cells (CSC), decreased senescence and eventual drug resistance phenotype. We interrogated some EMT markers that have been implicated in primary and bone metastasis of PCa using archived patient samples. Using an immunohistochemical approach, E-cadherin, Vimentin, ZEB1, Notch-1, PDGF-D and NF-κB were analyzed. Cases were microscopically scored using intensity (0, +1, +2, +3) and percentage of positive cells. Data was statistically analyzed using Fisher's Exact Test. Aberrant expression of EMT markers E-cadherin, Vimentin, PDGF-D, NF-κB, Notch-1 and ZEB1 was observed in PCa (primary tumor specimen) and bone metastasis tissues. The aberrant expression pattern varied according to the location within the tumor with higher expression was observed more at the invasive tumor front (ITF) vs. the center of the tumor. Notch-1 was significantly over-expressed in bone metastasis compared to primary PCa (p=0.057). The expression levels, intensity and % of positive cells of the remaining markers were not statistically significant in PCa vs. bone metastasis. In conclusion, protein expression analysis revealed the existence of EMT phenotype in the PCa and bone metastasis. Variation in the aberrant expression patterns at the invasive tumor front indicates the role of EMT markers in tumor invasion. Our results suggest that Notch-1 could play a role in PCa bone metastasis. Studies in larger patient cohorts are warranted before these EMT molecular markers can be translated to the clinical use.

The platelet-derived growth factor (PDGF) family was for more than 25 years assumed to consist of only PDGF-A and -B. The discovery of the novel family members PDGF-C and PDGF-D triggered a search for novel activities and complementary fine tuning between the members of this family of growth factors. Since the expansion of the PDGF family, more than 60 publications on the novel PDGF-C and PDGF-D have been presented, highlighting similarities and differences to the classical PDGFs. In this paper we review the published data on the PDGF family covering structural (gene and protein) similarities and differences among all four family members, with special focus on PDGF-C and PDGF-D expression and functions. Little information on the protein structures of PDGF-C and -D is currently available, but the PDGF-C protein may be structurally more similar to VEGF-A than to PDGF-B. PDGF-C contributes to normal development of the heart, ear, central nervous system (CNS), and kidney, while PDGF-D is active in the development of the kidney, eye and brain. In adults, PDGF-C is active in the kidney and the central nervous system. PDGF-D also plays a role in the lung and in periodontal mineralization. PDGF-C is expressed in Ewing family sarcoma and PDGF-D is linked to lung, prostate and ovarian cancers. Both PDGF-C and -D play a role in progressive renal disease, glioblastoma/medulloblastoma and fibrosis in several organs.

We describe the integration of microfluidic selection with high-throughput DNA sequencing technology for rapid and efficient discovery of nucleic acid aptamers. The Quantitative Selection of Aptamers through Sequencing method tracks the copy number and enrichment-fold of more than 10 million individual sequences through multiple selection rounds, enabling the identification of high-affinity aptamers without the need for the pool to fully converge to a small number of sequences. Importantly, this method allows the discrimination of sequences that arise from experimental biases rather than true high-affinity target binding. As a demonstration, we have identified aptamers that specifically bind to PDGF-BB protein with K(d) < 3 nM within 3 rounds. Furthermore, we show that the aptamers identified by Quantitative Selection of Aptamers through Sequencing have approximately 3-8-fold higher affinity and approximately 2-4-fold higher specificity relative to those discovered through conventional cloning methods. Given that many biocombinatorial libraries are encoded with nucleic acids, we extrapolate that our method may be extended to other types of libraries for a range of molecular functions.

OBJECTIVE

To determine the clinical efficacy of imatinib in patients with advanced aggressive fibromatosis (AF) and to identify the molecular basis of response/nonresponse to this agent.

METHODS

Nineteen patients with AF were treated with imatinib (800 mg/d) as part of a phase II clinical study. Tumor specimens were analyzed for mutations of KIT, PDGFRA, PDGFRB, and CTNNB1 (beta-catenin). Tumor expression of total and activated KIT, PDGFRA, and PDGFRB were assessed using immunohistochemistry and immunoblotting techniques. We also measured plasma levels of PDGF-AA and PDGF-BB in patients and normal patient controls.

RESULTS

Three of 19 patients (15.7%) had a partial response to treatment, with four additional patients having stable disease that lasted more than 1 year (overall 1 year tumor control rate of 36.8%). No mutations of KIT, PDGFRA, or PDGFRB were found. Sixteen of 19 patients (84%) had mutations involving the WNT pathway (APC or CTNNB1). However, there was no correlation between WNT pathway mutations and clinical response to imatinib. AF tumors expressed minimal to null levels of KIT and PDGFRA but expressed levels of PDGFRB that are comparable with normal fibroblasts. However, PDGFRB phosphorylation was not detected, suggesting that PDGFRB is only weakly activated. AF patients had elevated levels of PDGF-AA and PDGF-BB compared with normal patient controls. Notably, the plasma level of PDGF-BB was inversely correlated with time to treatment failure.

CONCLUSIONS

Imatinib is an active agent in the treatment of advanced AF. Imatinib response in AF patients may be mediated by inhibition of PDGFRB kinase activity.

A consistent response to liver injury is the activation of resident mesenchymal cells known as lipocytes (Ito, fat-storing cells) into a proliferating cell type. In cultured lipocytes, platelet-derived growth factor (PDGF) is the most potent proliferative cytokine, but requires the activation-dependent expression of its receptor protein (Friedman, S. L., and M. J. P. Arthur. 1989. J. Clin. Invest. 84:1780-1785); the role of PDGF receptor (PDGFR) in liver injury is unknown. We have examined PDGFR gene expression in freshly isolated lipocytes during liver injury and correlated these findings with a culture model of cellular activation. Whereas lipocytes from normal rats had no detectable transcript for the beta-PDGFR subunit, this mRNA was induced within 1 h after a dose of carbon tetrachloride (CCl4). In contrast, alpha subunit mRNA was detected in normal cells, but was unchanged after liver injury. Similar results were observed in lipocytes from bile duct-obstructed rats, although beta-PDGFR induction was less marked. By immunoblot, induction of beta-PDGFR protein in lipocytes isolated from CCl4-treated animals correlated with mRNA increases. In contrast to lipocytes, endothelial cells from normal liver expressed low levels of alpha- and beta-receptor subunit mRNA, which did not increase with injury. Using a beta-PDGFR antibody, receptor protein could be identified within fibrotic septa in CCl4-treated animals in regions where cells expressed proliferating cell nuclear antigen (PCNA). In cultured lipocytes activated by growth on uncoated plastic, beta-PDGFR transcripts appeared within 3 d after plating, which coincided with the onset of cellular proliferation. In contrast, quiescent cells in suspension culture had no detectable beta-PDGFR mRNA. These results indicate that beta-PDGF receptor induction by lipocytes is an early event during hepatic injury in vivo and in primary culture.

Abnormal vascular smooth muscle cell (SMC) proliferation and migration contribute to the development of restenosis after percutaneous transluminal coronary angioplasty and accelerated arteriopathy after cardiac transplantation. Previously, we reported that the macrolide antibiotic rapamycin, but not the related compound FK506, inhibits both human and rat aortic SMC proliferation in vitro by inhibiting cell cycle-dependent kinases and delaying phosphorylation of retinoblastoma protein (Marx, S.O., T. Jayaraman, L.O. Go, and A.R. Marks. 1995. Circ. Res. 362:801). In the present study the effects of rapamycin on SMC migration were assayed in vitro using a modified Boyden chamber and in vivo using a porcine aortic SMC explant model. Pretreatment with rapamycin (2 ng/ml) for 48 h inhibited PDGF-induced migration (PDGF BB homodimer; 20 ng/ml) in cultured rat and human SMC (n = 10; P < 0.0001), whereas FK506 had no significant effect on migration. Rapamycin administered orally (1 mg/kg per d for 7 d) significantly inhibited porcine aortic SMC migration compared with control (n = 15; P < 0.0001). Thus, in addition to being a potent immunosuppressant and antiproliferative, rapamycin also inhibits SMC migration.

The majority of human malignancies are believed to have epithelial origin, and the progression of cancer is often associated with a transient process named epithelial-mesenchymal transition (EMT). EMT is characterized by the loss of epithelial markers and the gain of mesenchymal markers that are typical of "cancer stem-like cells," which results in increased cell invasion and metastasis in vivo. Therefore, it is important to uncover the mechanistic role of factors that may induce EMT in cancer progression. Studies have shown that platelet-derived growth factor (PDGF) signaling contributes to EMT, and more recently, PDGF-D has been shown to regulate cancer cell invasion and angiogenesis. However, the mechanism by which PDGF-D promotes invasion and metastases and whether it is due to the acquisition of EMT phenotype remain elusive. For this study, we established stably transfected PC3 cells expressing high levels of PDGF-D, which resulted in the significant induction of EMT as shown by changes in cellular morphology concomitant with the loss of E-cadherin and zonula occludens-1 and gain of vimentin. We also found activation of mammalian target of rapamycin and nuclear factor-kappaB, as well as Bcl-2 overexpression, in PDGF-D PC3 cells, which was associated with enhanced adhesive and invasive behaviors. More importantly, PDGF-D-overexpressing PC3 cells showed tumor growth in SCID mice much more rapidly than PC3 cells. These results provided a novel mechanism by which PDGF-D promotes EMT, which in turn increases tumor growth, and these results further suggest that PDGF-D could be a novel therapeutic target for the prevention and/or treatment of prostate cancer. Disclosure of potential conflicts of interest is found at the end of this article.

Primary culture of rat brain endothelial cells is described, based on the method of C. C. W. Hughes and P. L. Lantos. The cells have been characterised using morphological and immunocytochemical techniques, and systematic studies undertaken to determine the optimal culture medium and conditions required to grow the cells at high purity on a variety of substrata. The endothelial cells have a spindle-shaped morphology, and proliferate as plaques from small clusters of cells associated with capillary fragments in the starting material. Tight junction-like cell:cell appositions are seen at the electron-microscopic level. The cells show characteristic staining for antigens recognized by antibodies against von Willebrand factor (Factor VIII-related antigen), angiotensin-converting enzyme (ACE), the transferrin receptor (Ox-26), actin and vimentin. They also show binding of the lectin from Ulex europaeus (UEA I). Potential contaminating cells include smooth muscle, fibroblasts, pericytes and meningeal cells. Contaminants can be kept to < ca. 5% by careful removal of large vessels and meninges during dissection, by brief treatment with Ca(2+)- and Mg(2+)-free saline, by growth in medium supplemented with plasma-derived serum treated for removal of platelet-derived growth factor (PDGF), and by occasional use of medium in which D-valine is substituted for L-valine. Cells attach well to collagen-coated plastic, less well to glass. Cells can be grown on transparent collagen filters (ICN, Cellagen and Costar, Transwell-Col), and on microcarrier beads (Pharmacia, Cytodex-3). The culture has proved to be a useful preparation for studies of cellular physiology, pharmacology and biochemistry of the brain endothelium, and represents a first step in producing an in vitro model of the rat blood-brain barrier.

Platelet-derived growth factor (PDGF), a smooth muscle cell (SMC) mitogen, and heparin-like glycosaminoglycans, known inhibitors of SMC growth and migration, were found to regulate thrombospondin synthesis and matrix deposition by cultured rat aortic SMC. The synthesis and distribution of thrombospondin was examined in growth-arrested SMCs, in PDGF-stimulated SMCs, and in heparin-treated SMCs using metabolic labeling and immunofluorescence techniques. Thrombospondin synthesis in response to purified PDGF occurred within 1 h after addition of growth factor to growth-arrested SMCs, peaked at 2 h, and returned to baseline levels by 5 h. The induction of synthesis of thrombospondin by PDGF was dose dependent, with a maximal effect observed at 2.5 ng/ml. Actinomycin D (2 micrograms/ml) inhibited thrombospondin induction by PDGF, suggesting a requirement for new RNA synthesis. In the presence of heparin and related polyanions, the incorporation of thrombospondin into the SMC extracellular matrix was markedly reduced. This effect was dose dependent with a maximal effect observed at a heparin concentration of 1 microgram/ml. Heparin did not affect the ability of SMCs to synthesize thrombospondin in response to PDGF. We interpret these data to suggest a role for thrombospondin in the SMC proliferative response to PDGF and in the regulation of SMC growth and migration by glycosaminoglycans.

Drosophila melanogaster hemocytes are highly motile macrophage-like cells that undergo a stereotypic pattern of migration to populate the whole embryo by late embryogenesis. We demonstrate that the migratory patterns of hemocytes at the embryonic ventral midline are orchestrated by chemotactic signals from the PDGF/VEGF ligands Pvf2 and -3 and that these directed migrations occur independently of phosphoinositide 3-kinase (PI3K) signaling. In contrast, using both laser ablation and a novel wounding assay that allows localized treatment with inhibitory drugs, we show that PI3K is essential for hemocyte chemotaxis toward wounds and that Pvf signals and PDGF/VEGF receptor expression are not required for this rapid chemotactic response. Our results demonstrate that at least two separate mechanisms operate in D. melanogaster embryos to direct hemocyte migration and show that although PI3K is crucial for hemocytes to sense a chemotactic gradient from a wound, it is not required to sense the growth factor signals that coordinate their developmental migrations along the ventral midline during embryogenesis.

Vascular smooth muscle cell (VSMC) proliferation and migration are responses to arterial injury that are highly important to the processes of restenosis and atherosclerosis. In the arterial balloon injury model in the rat, platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) are induced in the vessel wall and regulate these VSMC activities. Novel insulin sensitizing agents, thiazolidinediones, have been demonstrated to inhibit insulin and epidermal growth factor-induced growth of VSMCs. We hypothesized that these agents might also inhibit the effect of PDGF and bFGF on cultured VSMCs and intimal hyperplasia in vivo. Troglitazone (1 microM), a member of the thiazolidinedione class, produced a near complete inhibition of both bFGF-induced DNA synthesis as measured by bromodeoxyuridine incorporation (6.5+/-3.9 vs. 17.6+/-4.3% cells labeled, P < 0.05) and c-fos induction. This effect was associated with an inhibition (by 73+/-4%, P < 0.01) by troglitazone of the transactivation of the serum response element, which regulates c-fos expression. Inhibition of c-fos induction by troglitazone appeared to occur via a blockade of the MAP kinase pathway at a point downstream of MAP kinase activation by MAP kinase kinase. At this dose, troglitazone also inhibited PDGF-BB-directed migration of VSMC (by 70+/-6%, P < 0.01). These in vitro effects were operative in vivo. Quantitative image analysis revealed that troglitazone-treated rats had 62% (P < 0.001) less neointima/media area ratio 14 d after balloon injury of the aorta compared with injured rats that received no troglitazone. These results suggest troglitazone is a potent inhibitor of VSMC proliferation and migration and, thus, may be a useful agent to prevent restenosis and possibly atherosclerosis.

In the present study, we have identified several proteins in Swiss 3T3 cells that are phosphorylated on tyrosine in response to platelet-derived growth factor (PDGF) and exhibit an unusual bell-shaped dose-response curve with a maximum at 5 ng/ml platelet-derived growth factor (PDGF). These proteins include two that are associated with focal adhesions, namely the focal adhesion kinase (p125FAK), a novel cytosolic tyrosine kinase, and paxillin. At low concentrations of PDGF (1-5 ng/ml), these proteins are the predominant tyrosine-phosphorylated species. At 30 ng/ml PDGF, however, there was no stimulation of their phosphorylation over control levels. In contrast, tyrosine phosphorylation of previously described substrates of the PDGF receptor tyrosine kinase, namely the p21ras GTPase-activating protein, p120, phosphatidyl inositol 3' kinase, and phospholipase C gamma exhibited sigmoidal dose-response curves with PDGF and were all efficiently phosphorylated on tyrosine at 30 ng/ml PDGF. Cytochalasin D, which disrupts the actin cytoskeleton, completely inhibited the tyrosine phosphorylation of p125FAK and paxillin by PDGF. Examination of the actin cytoskeleton after stimulation of cells with different concentrations of PDGF revealed that at 5 ng/ml PDGF, actin appears in stress fibers and in membrane ruffles, while at 30 ng/ml, PDGF disrupts the actin cytoskeleton. Bombesin stimulates actin stress fiber formation with no evidence of disruption of stress fibers at high concentrations. When cells were stimulated with bombesin (10 nM) in the presence of 30 ng/ml PDGF, however, the actin cytoskeleton was completely disrupted. Further, the tyrosine phosphorylation of both p125FAK and paxillin induced by bombesin (10 nM) was completely prevented when cells were stimulated with bombesin in the presence of 30 ng/ml PDGF. We propose that the inhibitory limb in the bell-shaped dose-response curve of PDGF and the novel cross-talk between PDGF and bombesin on tyrosine phosphorylation may be explained by the ability of PDGF at 30 ng/ml to disrupt the actin cytoskeleton.

Normal blood microvessels are lined by pericytes, which contribute to microvessel development and stability through mechanisms that are poorly understood. Pericyte deficiency has been implicated in the pathogenesis of microvascular abnormalities associated with diabetes and tumors. However, the unambiguous identification of pericytes is still a problem because of cellular heterogeneity and few available molecular markers. Here we describe an approach to identify pericyte markers based on transcription profiling of pericyte-deficient brain microvessels isolated from platelet-derived growth factor (PDGF-B)-/- and PDGF beta receptor (PDGFRbeta)-/- mouse mutants. The approach was validated by the identification of known pericyte markers among the most down-regulated genes in PDGF-B-/- and PDGFRbeta-/- microvessels. Of candidates for novel pericyte markers, we selected ATP-sensitive potassium-channel Kir6.1 (also known as Kcnj8) and sulfonylurea receptor 2, (SUR2, also known as Abcc9), both part of the same channel complex, as well as delta homologue 1 (DLK1) for in situ hybridization, which demonstrated their specific expression in brain pericytes of mouse embryos. We also show that Kir6.1 is highly expressed in pericytes in brain but undetectable in pericytes in skin and heart. The three new brain pericyte markers are signaling molecules implicated in ion transport and intercellular signaling, potentially opening new windows on pericyte function in brain microvessels.

We investigated the interaction of phospholipase C-gamma (PLC-gamma) with wild-type and mutant forms of the platelet-derived growth factor (PDGF) beta-receptor both in vivo and in vitro. After PDGF treatment of CHO cell lines expressing wild-type or either of two mutant (delta Ki and Y825F) PDGF receptors, PLC-gamma became tyrosine phosphorylated and associated with the receptor proteins. The receptor association and tyrosine phosphorylation of PLC-gamma correlated with the ability of these receptors to mediate ligand-induced phosphatidylinositol turnover. However, both the delta Ki and Y825F mutant receptors were deficient in transmitting mitogenic signals, suggesting that the PDGF-induced tyrosine phosphorylation and receptor association of PLC-gamma are not sufficient to account for the growth-stimulatory activity of PDGF. Wild-type and delta Ki mutant PDGF receptor proteins expressed with recombinant baculovirus vectors also associated in vitro with mammalian PLC-gamma. However, baculovirus-expressed c-fms, v-fms, c-src, and Raf-1 proteins failed to associate with PLC-gamma under similar conditions. Phosphatase treatment of the baculovirus-expressed PDGF receptor greatly decreased its association with PLC-gamma. This requirement for receptor phosphorylation was also observed in vivo, where PLC-gamma could not associate with a mutant PDGF receptor (K602A) defective in autophosphorylation. PLC-gamma also coimmunoprecipitated with two other putative receptor substrates, the serine-threonine kinase Raf-1 and the 85-kilodalton phosphatidylinositol-3' kinase, presumably through its association with the ligand-activated receptor. Furthermore, baculovirus-expressed Raf-1 phosphorylated purified PLC-gamma in vitro at sites which showed increased serine phosphorylation in vivo in response to PDGF. These results suggest that PDGF directly influences PLC activity by inducing the association of PLC-gamma with a receptor signaling complex, resulting in increased tyrosine and serine phosphorylation of PLC-gamma.

The low density lipoprotein (LDL) receptor gene family represents a class of multifunctional, endocytic cell surface receptors. Recently, roles in cellular signaling have also emerged. For instance, the very low density lipoprotein receptor (VLDLR) and the apolipoprotein receptor-2 (apoER2) function in a developmental signaling pathway that regulates the lamination of cortical layers in the brain and involves the activation of tyrosine kinases. Furthermore, the cytoplasmic domain of the LDL receptor-related protein (LRP) was found to be a substrate for the non-receptor tyrosine kinase Src, but the physiological significance of this phosphorylation event remained unknown. Here we show that tyrosine phosphorylation of LRP occurs in caveolae and involves the platelet-derived growth factor (PDGF) receptor beta and phosphoinositide 3-kinase. Receptor-associated protein, an antagonist of ligand binding to LRP, and apoE-enriched beta-VLDL, a ligand for LRP, reduce PDGF-induced tyrosine phosphorylation of the LRP cytoplasmic domain. In the accompanying paper (Loukinova, E., Ranganathan, S., Kuznetsov, S., Gorlatova, N., Migliorini, M., Ulery, P. G., Mikhailenko, I., Lawrence, D. L., and Strickland, D. K. (2002) J. Biol. Chem. 277, 15499-15506) Loukinova et al. further demonstrate that one form of PDGF, PDGF-BB, binds specifically to LRP and that phosphorylation of LRP requires the activation of Src family kinases. Taken together, these findings provide a biochemical basis for a cellular signaling pathway that involves apoE and LRP.

Insulin-dependent phosphorylation of Akt target AS160 is required for GLUT4 translocation. Insulin and platelet-derived growth factor (PDGF) (Akt activators) or activation of conventional/novel (c/n) protein kinase C (PKC) and 5' AMP-activated protein kinase (AMPK) all promote a rise in membrane GLUT4 in skeletal muscle and cultured cells. However, the downstream effectors linking these pathways to GLUT4 traffic are unknown. Here we explore the hypothesis that AS160 is a molecular link among diverse signaling cascades converging on GLUT4 translocation. PDGF and insulin increased AS160 phosphorylation in CHO-IR cells. Stimuli that activate c/n PKC or AMPK also elevated AS160 phosphorylation. We therefore examined if these signaling pathways engage AS160 to regulate GLUT4 traffic in muscle cells. Nonphosphorylatable AS160 (4P-AS160) virtually abolished the net surface GLUT4myc gains elicited by insulin, PDGF, K(+) depolarization, or 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside but partly, yet significantly, inhibited the effects of 4-phorbol-12-myristate-13-acetate. However, the hypertonicity or 2,4-dinitrophenol-dependent gains in surface GLUT4myc were unaffected by 4P-AS160. RK-AS160 (GTPase-activating protein [GAP] inactive) or 4PRK-AS160 (GAP inactive, nonphosphorylatable) had no effect on surface GLUT4myc elicited by all stimuli. Collectively, these results indicate that activation of Akt, c/n PKC, or alpha2-AMPK intersect at AS160 to regulate GLUT4 traffic, as well as highlight the potential of AS160 as a therapy target to increase muscle glucose uptake.

Abnormal expression of human amyloid precursor protein (hAPP) gene products may play a critical role in Alzheimer's disease (AD). Recently, a transgenic model was established in which platelet-derived growth factor (PDGF) promoter-driven neuronal expression of an alternatively spliced hAPP minigene resulted in prominent AD-type neuropathology (Games, D., Adams, D., Alessandrini, R., Barbour, R., Berthelette, P., Blackwell, C., Carr, T., Clemens, J., Donaldson, T., Gillespie, F., Guido, T., Hagopian, S., Johnson-Wood, K., Khan, K., Lee, M., Leibowitz, P., Lieberburg, I., Little, S., Masliah, E., McConlogue, L., Montoya-Zavala, M., Mucke, L., Paganini, L., and Penniman, E. (1995) Nature 373, 523-527). Here we compared the levels and alternative splicing of APP transcripts in brain tissue of hAPP transgenic and nontransgenic mice and of humans with and without AD. PDGF-hAPP mice showed severalfold higher levels of total APP mRNA than did nontransgenic mice or humans, whereas their endogenous mouse APP mRNA levels were decreased. This resulted in a high ratio of mRNAs encoding mutated hAPP versus wild-type mouse APP. Modifications of hAPP introns 6, 7, and 8 in the PDGF-hAPP construct resulted in a prominent change in alternative splice site selection with transcripts encoding hAPP770 or hAPP751 being expressed at substantially higher levels than hAPP695 mRNA. Frontal cortex of humans with AD showed a subtle increase in the relative abundance of hAPP751 mRNA compared with normal controls. These data identify specific intron sequences that may contribute to the normal neuronspecific alternative splicing of APP pre-mRNA in vivo and support a causal role of hAPP gene products in the development of AD-type brain alterations.

Human mononuclear cells were plated in culture, and the conditioned media of these cells were analyzed by heparin-Sepharose affinity chromatography. The fractions were tested for growth factor activity as measured by the stimulation of DNA synthesis in BALB/c 3T3 cells. After 2 d in culture, two peaks of heparin-binding growth factor (HBGF) activity were detected, one eluting with 0.5 M NaCl, which could be shown to be platelet-derived growth factor (PDGF)-like, and the other eluting with 1.0 M NaCl. After 7-11 d in culture, when monocytes had clearly differentiated into macrophages, greater than 95% of the HBGF activity in conditioned medium consisted of the 1.0 M NaCl elution peak. This activity, which was designated macrophage-derived HBGF (MD-HBGF), was found to be a cationic heat-resistant polypeptide with a molecular weight in the range of 14-25 kDa. Analysis using Western blots and specific neutralizing antisera, as well as comparative heparin affinity analysis, indicated that MD-HBGF was not identical to other heparin-binding 3T3 cell growth factors known to be produced by macrophages, such as PDGF (AB, AA, and BB forms), acidic fibroblast growth factor, and basic fibroblast growth factor. In addition to stimulating mitogenesis in 3T3 cells, MD-HBGF also stimulated the proliferation of vascular smooth muscle cells, but did not stimulate the proliferation of vascular endothelial cells.

Global inhibition of class I and II histone deacetylases (HDACs) impairs angiogenesis. Herein, we have undertaken the identification of the specific HDAC(s) with activity that is necessary for the development of blood vessels. Using small interfering RNAs, we observed that HDAC7 silencing in endothelial cells altered their morphology, their migration, and their capacity to form capillary tube-like structures in vitro but did not affect cell adhesion, proliferation, or apoptosis. Among several factors known to be involved in angiogenesis, platelet-derived growth factor-B (PDGF-B) and its receptor (PDGFR-beta) were the most upregulated genes following HDAC7 silencing. We demonstrated that their increased expression induced by HDAC7 silencing was partially responsible for the inhibition of endothelial cell migration. In addition, we have also shown that treatment of endothelial cells with phorbol 12-myristate 13-acetate resulted in the exportation of HDAC7 out of the nucleus through a protein kinase C/protein kinase D activation pathway and induced, similarly to HDAC7 silencing, an increase in PDGF-B expression, as well as a partial inhibition of endothelial cell migration. Collectively, these data identified HDAC7 as a key modulator of endothelial cell migration and hence angiogenesis, at least in part, by regulating PDGF-B/PDGFR-beta gene expression. Because angiogenesis is required for tumor progression, HDAC7 may represent a rational target for therapeutic intervention against cancer.

Hepatic lipocytes are perisinusoidal cells that have been thought to be analogous to tissue pericytes, a cell type with purported vasoregulatory properties. However, we and others have recently demonstrated that lipocytes acquire markers of smooth muscle cells or myofibroblasts only after liver injury, via a process termed "activation." In this study, we document lipocyte contractility on collagen lattices and examine the importance of activation in this process. In culture, lipocytes became contractile only after spreading and activating, coincident with expression of smooth muscle alpha actin, a marker of activation (1990. Virchows Arch. B Cell Pathol. 59:349). After 5 d in culture, lipocytes induced rapid and sustained contraction of collagen lattices (to 43.7 +/- 2.3% of their original size 24 h after detachment). There was no contraction of lattices containing hepatocytes. Scanning electron microscopy demonstrated intimate associations of lipocyte cell membranes and collagen fibrils. Reduction in cell volume during contraction was also prominent. Lattice contraction by lipocytes was proportional to cell number. Serum was a potent stimulator of lipocyte contraction, as were endothelin types 1, 2, and 3; the effect of serum and endothelin 1 were additive. Neither thrombin, angiotensin-II, serotonin, nor the cytokines PDGF and TGF beta induced contraction. Cytochalasin B treatment resulted in concentration-dependent inhibition of contraction. As a test of the in vivo relevance of the culture findings, lipocytes were isolated from fibrotic animals and examined immediately after adherence. Whereas lipocytes from normal liver were initially compact, smooth muscle alpha actin negative and noncontractile, cells from animals with hepatic injury due to CCl4 displayed an activated appearance, expressed smooth muscle alpha actin, and were contractile immediately after adherence. Additionally, IFN-gamma, an agent which blocks lipocyte activation (1992. Hepatology. 16:776), inhibited lipocyte contraction. The data document that normal (i.e., quiescent) lipocytes are not contractile, but that activation is associated with the development of contractility. These findings suggest that a role for lipocytes in organ contraction or vasoregulation may be confined to injured, not normal liver.

We have isolated Swiss 3T3 subclones that are resistant to the mitogenic and morphological transforming effects of v-Src as a consequence of aberrant translocation of the oncoprotein under low serum conditions. In chicken embryo and NIH 3T3 fibroblasts under similar conditions, v-Src rapidly translocates from the perinuclear region to the focal adhesions upon activation of the tyrosine kinase, resulting in downstream activation of activator protein-1 and mitogen-activated protein kinase, which are required for the mitogenic and transforming activity of the oncoprotein. Since serum deprivation induces cytoskeletal disorganization in Swiss 3T3, we examined whether regulators of the cytoskeleton play a role in the translocation of v-Src, and also c-Src, in response to biological stimuli. Actin stress fibers and translocation of active v-Src to focal adhesions in quiescent Swiss 3T3 cells were restored by microinjection of activated Rho A and by serum. Double labeling with anti-Src and phalloidin demonstrated that v-Src localized along the reformed actin filaments in a pattern that would be consistent with trafficking in complexes along the stress fibers to focal adhesions. Furthermore, treatment with the actin-disrupting drug cytochalasin D, but not the microtubule-disrupting drug nocodazole, prevented v-Src translocation. In addition to v-Src, we observed that PDGF-induced, Rac-mediated membrane ruffling was accompanied by translocation of c-Src from the cytoplasm to the plasma membrane, an effect that was also blocked by cytochalasin D. Thus, we conclude that translocation of Src from its site of synthesis to its site of action at the cell membrane requires an intact cytoskeletal network and that the small G proteins of the Rho family may specify the peripheral localization in focal adhesions or along the membrane, mediated by their effects on the cytoskeleton.

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is an endothelial cell-specific mitogen that is structurally related to platelet-derived growth factor (PDGF). Vascular endothelial growth factor/vascular permeability factor induces angiogenesis in vivo and may play a critical role in tumor angiogenesis. Using immunohistochemical analysis, the authors demonstrated the presence of VEGF/VPF protein in surgical specimens of glioblastoma multiforme and cultured glioma cells. By means of an enzyme-linked immunosorbent assay (ELISA) of cell supernatants, the authors showed that VEGF/VPF is variably secreted by all nine cultured human malignant glioma cell lines (CH-235MG, D-37MG, D-54MG, D-65MG, U-87MG, U-105MG, U-138MG, U-251MG, U-373MG) and by a single meningioma cell line (CH-157MN). An immunocytochemical survey of these cell lines revealed a cytoplasmic and cell-surface distribution of VEGF/VPF. In the U-105MG glioma cell line, VEGF/VPF secretion was induced with physiological concentrations of epidermal growth factor, PDGF-BB, or basic fibroblast growth factor, but not with PDGF-AA. Moreover, it was observed that activation of convergent growth factor signaling pathways led to increased glioma VEGF secretion. Similar results were obtained using these growth factor combinations in the D-54MG glioma cell line. The data obtained suggest a potential role for VEGF/VPF in tumor hypervascularity and peritumoral edema. These observations may lead to development of new therapeutic strategies.