Tumour cell CD99 regulates transendothelial migration via CDC42 and actin remodelling.

Metastasis requires tumour cells to cross endothelial cell (EC) barriers using pathways similar to those used by leucocytes during inflammation. Cell surface CD99 is expressed by healthy leucocytes and ECs, and participates in inflammatory transendothelial migration (TEM). Tumour cells also express CD99, and we have analysed its role in tumour progression and cancer cell TEM. Tumour cell CD99 was required for adhesion to ECs but inhibited invasion of the endothelial barrier and migratory activity. Furthermore, CD99 depletion in tumour cells caused redistribution of the actin cytoskeleton and increased activity of the Rho GTPase CDC42, known for its role in actin remodelling and cell migration. In a xenograft model of breast cancer, tumour cell CD99 expression inhibited metastatic progression, and patient samples showed reduced expression of the CD99 gene in brain metastases compared to matched primary breast tumours. We conclude that CD99 negatively regulates CDC42 and cell migration. However, CD99 has both pro- and anti-tumour activity, and our data suggest that this results in part from its functional linkage to CDC42 and the diverse signalling pathways downstream of this Rho GTPase. This article has an associated First Person interview with the first author of the paper.

Acute aerobic exercise-conditioned serum reduces colon cancer cell proliferation in vitro through interleukin-6-induced regulation of DNA damage.

Epidemiological evidence shows that regular physical activity is associated with reduced risk of primary and recurrent colon cancer. However, the underlying mechanisms of action are poorly understood. We evaluated the effects of stimulating a human colon cancer cell line (LoVo) with human serum collected before and after an acute exercise bout vs nonexercise control serum on cancer cell proliferation. We also measured exercise-induced changes in serum cytokines and intracellular protein expression to explore potential biological mechanisms. Blood samples were collected from 16 men with lifestyle risk factors for colon cancer (age ≥50 years; body mass index ≥25 kg/m2 ; physically inactive) before and immediately after an acute bout of moderate-intensity aerobic interval exercise (6 × 5 minutes intervals at 60% heart rate reserve) and a nonexercise control condition. Stimulating LoVo cells with serum obtained immediately after exercise reduced cancer cell proliferation compared to control (-5.7%; P = .002). This was accompanied by a decrease in LoVo cell γ-H2AX expression (-24.6%; P = .029), indicating a reduction in DNA damage. Acute exercise also increased serum IL-6 (24.6%, P = .002). Furthermore, stimulating LoVo cells with recombinant IL-6 reduced γ-H2AX expression (ß = -22.7%; P < .001) and cell proliferation (ß = -5.3%; P < .001) in a linear dose-dependent manner, mimicking the effect of exercise. These findings suggest that the systemic responses to acute aerobic exercise inhibit colon cancer cell proliferation in vitro, and this may be driven by IL-6-induced regulation of DNA damage and repair. This mechanism of action may partly underlie epidemiological associations linking regular physical activity with reduced colon cancer risk.

A novel p53 mutant found in iatrogenic urothelial cancers is dysfunctional and can be rescued by a second-site global suppressor mutation.

Exposure to herbal remedies containing the carcinogen aristolochic acid (AA) has been widespread in some regions of the world. Rare A→T TP53 mutations were recently discovered in AA-associated urothelial cancers. The near absence of these mutations among all other sequenced human tumors suggests that they could be biologically silent. There are no cell banks with established lines derived from human tumors with which to explore the influence of the novel mutants on p53 function and cellular behavior. To investigate their impact, we generated isogenic mutant clones by integrase-mediated cassette exchange at the p53 locus of platform (null) murine embryonic fibroblasts and kidney epithelial cells. Common tumor mutants (R248W, R273C) were compared with the AA-associated mutants N131Y, R249W, and Q104L. Assays of cell proliferation, migration, growth in soft agar, apoptosis, senescence, and gene expression revealed contrasting outcomes on cellular behavior following introduction of N131Y or Q104L. The N131Y mutant demonstrated a phenotype akin to common tumor mutants, whereas Q104L clone behavior resembled that of cells with wild-type p53. Wild-type p53 responses were restored in double-mutant cells harboring N131Y and N239Y, a second-site rescue mutation, suggesting that pharmaceutical reactivation of p53 function in tumors expressing N131Y could have therapeutic benefit. N131Y is likely to contribute directly to tumor phenotype and is a promising candidate biomarker of AA exposure and disease. Rare mutations thus do not necessarily point to sites where amino acid exchanges are phenotypically neutral. Encounter with mutagenic insults targeting cryptic sites can reveal specific signature hotspots.

Expression of biologically active human colony stimulating factor-1 in Pichia pastoris.

Colony Stimulating Factor-1 (CSF-1) is involved in proliferation, differentiation, and survival of the mononuclear lineage, in development of the female reproductive system and mammary glands during pregnancy and lactation. It is also implicated in the biology of breast cancer and promotion of its metastasis to bones. Therefore, CSF-1 is required for many applications in cellular and molecular biology studies. Commercial products, usually expressed in prokaryotic systems, are costly, with the likelihood of endotoxin contamination and also lack posttranslational modifications. These considerations provide the rationale to express growth factors in eukaryotic systems. In this study, the biologically active and soluble fragment (residues 33-182) of human (CSF-1) was cloned from K562 cell line and expressed in Pichia pastoris. The expression level of the active CSF-1 was about 100 µg/ml of the P. pastoris culture medium. Protein analysis revealed that the expressed CSF-1 appears in three bands with apparent molecular weight of 30, 26 and 20 kDa constituting 44%, 25% and 13% of all proteins in the culture medium, respectively. The expressed protein was partially purified and concentrated (10x) by ultrafiltration, then filter sterilized. The product was confirmed to be biologically active by stimulation of its receptor (FMS) autophosphorylation in THP-1 cells and also growth promotion of factor dependent FDC-P1 cells expressing human wild-type FMS (FD-FMS-WT). Therefore, P. pastoris is a highly efficient and cost-effective expression system for production of endotoxin-free CSF-1 for research and potentially for therapeutic applications.

A biphasic endothelial stress-survival mechanism regulates the cellular response to vascular endothelial growth factor A.

Vascular endothelial growth factor A (VEGF-A) is an essential cytokine that regulates endothelial function and angiogenesis. VEGF-A binding to endothelial receptor tyrosine kinases such as VEGFR1 and VEGFR2 triggers cellular responses including survival, proliferation and new blood vessel sprouting. Increased levels of a soluble VEGFR1 splice variant (sFlt-1) correlate with endothelial dysfunction in pathologies such as pre-eclampsia; however the cellular mechanism(s) underlying the regulation and function of sFlt-1 are unclear. Here, we demonstrate the existence of a biphasic stress response in endothelial cells, using serum deprivation as a model of endothelial dysfunction. The early phase is characterized by a high VEGFR2:sFlt-1 ratio, which is reversed in the late phase. A functional consequence is a short-term increase in VEGF-A-stimulated intracellular signaling. In the late phase, sFlt-1 is secreted and deposited at the extracellular matrix. We hypothesized that under stress, increased endothelial sFlt-1 levels reduce VEGF-A bioavailability: VEGF-A treatment induces sFlt-1 expression at the cell surface and VEGF-A silencing inhibits sFlt-1 anchorage to the extracellular matrix. Treatment with recombinant sFlt-1 inhibits VEGF-A-stimulated in vitro angiogenesis and sFlt-1 silencing enhances this process. In this response, increased VEGFR2 levels are regulated by the phosphatidylinositol-3-kinase and PKB/Akt signaling pathways and increased sFlt-1 levels by the ERK1/2 signaling pathway. We conclude that during serum withdrawal, cellular sensing of environmental stress modulates sFlt-1 and VEGFR2 levels, regulating VEGF-A bioavailability and ensuring cell survival takes precedence over cell proliferation and migration. These findings may underpin an important mechanism contributing to endothelial dysfunction in pathological states.

A research-led flexible cell biology practical for biological sciences undergraduate and postgraduate degree courses.

A challenge in the pandemic era is to implement effective but flexible practical teaching for biological sciences courses. Such teaching needs to deliver conceptual, analytical and practical skills training while having the option to rapidly respond to health and safety issues, local regulations, staff and student concerns. In this paper, we describe a set of cell biology practicals (mini-project) that meets many of these requirements and provides flexibility in providing skills training both through online and in practical laboratory environments. We have used a human adenocarcinoma cell line A431 stably transfected with a fluorescent cell cycle reporter as a biological model to deliver training through discrete work packages encompassing cell culture, fluorescence microscopy, biochemistry and statistics. How such work packages can be modified to, an online format either partially or completely is also described. Furthermore, the activities can be adapted for teaching both undergraduate and postgraduate level courses to ensure effective skills training which is applicable to a wide range of biological degree programs and levels of study.

Pro- and anti-tumour activities of CD146/MCAM in breast cancer result from its heterogeneous expression and association with epithelial to mesenchymal transition.

CD146, also known as melanoma cell adhesion molecule (MCAM), is expressed in numerous cancers and has been implicated in the regulation of metastasis. We show that CD146 negatively regulates transendothelial migration (TEM) in breast cancer. This inhibitory activity is reflected by a reduction in MCAM gene expression and increased promoter methylation in tumour tissue compared to normal breast tissue. However, increased CD146/MCAM expression is associated with poor prognosis in breast cancer, a characteristic that is difficult to reconcile with inhibition of TEM by CD146 and its epigenetic silencing. Single cell transcriptome data revealed MCAM expression in multiple cell types, including the malignant cells, tumour vasculature and normal epithelium. MCAM expressing malignant cells were in the minority and expression was associated with epithelial to mesenchymal transition (EMT). Furthermore, gene expression signatures defining invasiveness and a stem cell-like phenotype were most strongly associated with mesenchymal-like tumour cells with low levels of MCAM mRNA, likely to represent a hybrid epithelial/mesenchymal (E/M) state. Our results show that high levels of MCAM gene expression are associated with poor prognosis in breast cancer because they reflect tumour vascularisation and high levels of EMT. We suggest that high levels of mesenchymal-like malignant cells reflect large populations of hybrid E/M cells and that low CD146 expression on these hybrid cells is permissive for TEM, aiding metastasis.

Ligand-stimulated VEGFR2 signaling is regulated by co-ordinated trafficking and proteolysis.

Vascular endothelial growth factor A (VEGF-A)-induced signaling through VEGF receptor 2 (VEGFR2) regulates both physiological and pathological angiogenesis in mammals. However, the temporal and spatial mechanism underlying VEGFR2-mediated intracellular signaling is not clear. Here, we define a pathway for VEGFR2 trafficking and proteolysis that regulates VEGF-A-stimulated signaling and endothelial cell migration. Ligand-stimulated VEGFR2 activation and ubiquitination preceded proteolysis and cytoplasmic domain removal associated with endosomes. A soluble VEGFR2 cytoplasmic domain fragment displayed tyrosine phosphorylation and activation of downstream intracellular signaling. Perturbation of endocytosis by the depletion of either clathrin heavy chain or an ESCRT-0 subunit caused differential effects on ligand-stimulated VEGFR2 proteolysis and signaling. This novel VEGFR2 proteolysis was blocked by the inhibitors of 26S proteasome activity. Inhibition of proteasome activity prolonged VEGF-A-induced intracellular signaling to c-Akt and endothelial nitric oxide synthase (eNOS). VEGF-A-stimulated endothelial cell migration was dependent on VEGFR2 and VEGFR tyrosine kinase activity. Inhibition of proteasome activity in this assay stimulated VEGF-A-mediated endothelial cell migration. VEGFR2 endocytosis, ubiquitination and proteolysis could also be stimulated by a protein kinase C-dependent pathway. Thus, removal of the VEGFR2 carboxyl terminus linked to phosphorylation, ubiquitination and trafficking is necessary for VEGF-stimulated endothelial signaling and cell migration.

In Vitro Co-culture of Fibroblast and Endothelial Cells to Assess Angiogenesis.

Angiogenesis relies on the spatial and temporal coordination of endothelial migration and proliferation to form new blood vessels. This occurs through synchronous activation of multiple downstream pathways which facilitate vascular development. Proangiogenic growth factors and supporting extracellular matrix allow the formation of capillary-like tubules, reminiscent of microvascular beds, in vitro. In this chapter, we describe a methodology for the establishment of vascular networks by co-culture of endothelial cells and fibroblasts to facilitate the study of tubulogenic and angiogenic potential. We detail the use of siRNA mediated knockdown to deplete target genes of interest, in either the endothelial or fibroblast cells, to allow the assessment of their role in angiogenesis. Finally, we detail how these vascular networks may be stained using immunofluorescence to allow quantification of angiogenic potential in vitro.

Wild-type and Hupki (human p53 knock-in) murine embryonic fibroblasts: p53/ARF pathway disruption in spontaneous escape from senescence.

Research on cell senescence and immortalization of murine embryonic fibroblasts (MEFs) has revealed important clues about genetic control of senescence in humans. To investigate senescence and genetic alterations in the p53 pathway that lead to senescence bypass in culture, we compared the behavior of MEFs from wild-type mice with MEFs from Hupki mice, which harbor a humanized p53 gene. We found that humanizing the p53 gene in mice preserved major features of the MEF senescence/immortalization process. In both genotypes, a significant proportion of spontaneously arising cell lines had sustained either a p53 point mutation or p19/ARF biallelic deletion. The p53 mutations selected for during Hupki MEF immortalization have been found in human tumors and are classified in the yeast transactivation assay as transcriptionally defunct, suggesting that disabling this component of p53 activity is crucial in senescence bypass. Surprisingly, in spontaneously immortalized cell lines from both wild-type and Hupki MEFs, the predominant type of p53 mutation was a G to C transversion, rather than the G to T substitutions expected from the raised oxygen levels characteristic of standard culture conditions. Over half of the cell lines did not reveal evidence of p53 mutation or loss of p19/ARF and retained a robust wild-type p53 response to DNA damage, supporting the inference from senescence bypass screens that alternative genetic routes to immortalization occur.

Rab GTPase regulation of VEGFR2 trafficking and signaling in endothelial cells.

OBJECTIVE: Vascular endothelial growth factor receptor 2 (VEGFR2) is a receptor tyrosine kinase that regulates vascular physiology. However, mechanism(s) by which VEGFR2 signaling and trafficking is coordinated are not clear. Here, we have tested endocytic Rab GTPases for regulation of VEGFR2 trafficking and signaling linked to endothelial cell migration. METHODS AND RESULTS: Quiescent VEGFR2 displays endosomal localization and colocalization with the Rab5a GTPase, an early endosome fusion regulator. Expression of GTP or GDP-bound Rab5a mutants block activated VEGFR2 trafficking and degradation. Manipulation of Rab7a GTPase activity associated with late endosomes using overexpression of wild-type or mutant proteins blocks activated VEGFR2 trafficking and degradation. Depletion of Rab7a decreased VEGFR2 Y1175 phosphorylation but increased p42/44 (pERK1/2) MAPK phosphorylation. Endothelial cell migration was increased by Rab5a depletion but decreased by Rab7a depletion. CONCLUSIONS: Rab5a and Rab7a regulate VEGFR2 trafficking toward early and late endosomes. Our data suggest that VEGFR2-mediated regulation of endothelial function is dependent on different but specific Rab-mediated GTP hydrolysis activity required for endosomal trafficking.

ATF-2 and Tpl2 regulation of endothelial cell cycle progression and apoptosis.

Cells respond to soluble and membrane-bound factors to activate signalling cascades that control cell proliferation and cell death. Vascular endothelial growth factor A (VEGF-A) is a soluble ligand that modulates a variety of cellular responses including cell proliferation and apoptosis. It is not well understood how VEGF-A signalling pathways regulate cell proliferation and cell death. To address this, we examined VEGF-A-regulated signalling pathways in the cytosol and nucleus and functional requirement for such cellular responses. The VEGF-A-regulated transcription factor, ATF-2, is required for cell cycle proteins such as p53, p21 and Cyclin D1. A cytosolic serine/threonine protein kinase (Tpl2) modulates ATF-2-regulated effects on the endothelial cell cycle. Such regulatory effects impact on endothelial cell proliferation, cell viability and apoptosis. These cellular effects influence complex cell-based organisation such as endothelial tubulogenesis. Our study now provides a framework for incorporating VEGF-A-stimulated signalling events from the cytosol to the nucleus which helps to understand how cell proliferation and apoptosis are controlled.

Tpl2 is required for VEGF-A-stimulated signal transduction and endothelial cell function.

New blood vessel sprouting (angiogenesis) and vascular physiology are fundamental features of metazoan species but we do not fully understand how signal transduction pathways regulate diverse vascular responses. The vascular endothelial growth factor (VEGF) family bind membrane-bound receptor tyrosine kinases (VEGFRs), which trigger multiple signal transduction pathways and diverse cellular responses. We evaluated whether the MAP3K family member and proto-oncoprotein Tpl2 (MAP3K8) regulates basal and VEGF-A-stimulated signal transduction in endothelial cells. Notably, stimulation with exogenous VEGF-A increased Tpl2 mRNA levels and consequently de novo protein synthesis. Depletion of Tpl2 levels reveals a role in both basal and VEGF-A-stimulated endothelial cell responses, including endothelial-leukocyte interactions, monolayer permeability and new blood vessel formation. Under basal conditions, Tpl2 modulates a signal transduction cascade resulting in phosphorylation of a nuclear transcription factor (ATF-2) and altered endothelial gene expression, a pathway previously identified as crucial in VEGF-dependent vascular responses. Loss of Tpl2 expression or activity impairs signal transduction through Akt, eNOS and ATF-2, broadly impacting on endothelial function. Our study now provides a mechanism for Tpl2 as a central component of signal transduction pathways in the endothelium.

Early myeloid cells expressing c-KIT isoforms differ in signal transduction, survival and chemotactic responses to Stem Cell Factor.

Isoforms of the receptor tyrosine kinase, c-KIT, differ in the presence or absence of a GNNK tetrapeptide in the extracellular juxtamembrane region. When expressed in murine NIH3T3 cells, these isoforms of c-KIT showed differential activation of signaling pathways and proliferation in response to Stem Cell Factor (SCF). However, c-KIT is not normally expressed by fibroblasts, but plays a key role in hematopoiesis. Because signaling pathways and cellular responses mediated by c-KIT differ in different cell types, we studied the effects of SCF stimulation on factor-dependent murine early myeloid cells expressing human GNNK+ or GNNK- c-KIT. As in fibroblasts, SCF activation of the GNNK- isoform resulted in stronger, more rapid receptor phosphorylation, and activation of Src kinases, while only a minor effect on the phosphatidylinositol 3-kinase pathway was observed. Similarly, more rapid Src kinase-dependent internalisation of the GNNK- isoform occurred in response to SCF. In contrast to fibroblasts, only minor differences in ERK activation were seen indicating that early hematopoietic cells, unlike fibroblasts, are not dependent on Src kinases for activation of this pathway in response to SCF. Enhanced SCF-dependent growth was observed in GNNK- c-KIT expressing cells due to lower cell attrition. The rate of cell division was similar. Importantly, cells expressing the GNNK- isoform showed a greater chemotactic response to SCF.

Resistance to c-KIT kinase inhibitors conferred by V654A mutation.

Certain mutations within c-KIT cause constitutive activation of the receptor and have been associated with several human malignancies. These include gastrointestinal stromal tumors (GIST), mastocytosis, acute myelogenous leukemia, and germ cell tumors. The kinase inhibitor imatinib potently inhibits c-KIT and is approved for treatment of GIST. However, secondary point mutations can develop within the kinase domain to confer resistance to imatinib and cause drug-resistant relapse. A common mutation, which results in a V654A substitution, has been documented in imatinib-resistant GIST patients. We expressed c-KIT cDNA constructs encoding the V654A substitution alone and in combination with a typical activating exon 11 mutation characteristic of GIST, V560G, in factor-dependent FDC-P1 cells. The V654A substitution alone resulted in enhanced proliferation in c-KIT ligand (stem cell factor) but not factor independence. Cells expressing the double mutant were, like those expressing single V560G mutant c-KIT, factor independent. Analysis of cellular proliferation in the presence of imatinib showed that the V654A substitution alone conferred resistance. The difference in sensitivity was especially pronounced for cells expressing single mutant V560G c-KIT compared with double mutant V560G/V654A c-KIT. The findings were supported by studies of c-KIT phosphorylation. Analysis of the crystal structure of imatinib in complex with the kinase domain of c-KIT predicts that the V654A substitution directly affects the binding of imatinib to the receptor. Alternative c-KIT inhibitors, nilotinib (AMN107) and PKC412, were also less active on V560G/V654A c-KIT than on the V560G single mutant; however, nilotinib, like imatinib, potently inhibited the V560G mutant. PKC412 strongly inhibited imatinib-resistant D816V c-KIT.

Genome-wide functional analysis reveals central signaling regulators of lymphatic endothelial cell migration and remodeling.

Lymphatic vessels constitute a specialized vasculature that is involved in development, cancer, obesity, and immune regulation. The migration of lymphatic endothelial cells (LECs) is critical for vessel growth (lymphangiogenesis) and vessel remodeling, processes that modify the lymphatic network in response to developmental or pathological demands. Using the publicly accessible results of our genome-wide siRNA screen, we characterized the migratome of primary human LECs and identified individual genes and signaling pathways that regulate LEC migration. We compared our data set with mRNA differential expression data from endothelial and stromal cells derived from two in vivo models of lymphatic vessel remodeling, viral infection and contact hypersensitivity-induced inflammation, which identified genes selectively involved in regulating LEC migration and remodeling. We also characterized the top candidates in the LEC migratome in primary blood vascular endothelial cells to identify genes with functions common to lymphatic and blood vascular endothelium. On the basis of these analyses, we showed that LGALS1, which encodes the glycan-binding protein Galectin-1, promoted lymphatic vascular growth in vitro and in vivo and contributed to maintenance of the lymphatic endothelial phenotype. Our results provide insight into the signaling networks that control lymphangiogenesis and lymphatic remodeling and potentially identify therapeutic targets and biomarkers in disease specific to lymphatic or blood vessels.

Vascular endothelial growth factor A-stimulated signaling from endosomes in primary endothelial cells.

The vascular endothelial growth factor A (VEGF-A) is a multifunctional cytokine that stimulates blood vessel sprouting, vascular repair, and regeneration. VEGF-A binds to VEGF receptor tyrosine kinases (VEGFRs) and stimulates intracellular signaling leading to changes in vascular physiology. An important aspect of this phenomenon is the spatiotemporal coordination of VEGFR trafficking and intracellular signaling to ensure that VEGFR residence in different organelles is linked to downstream cellular outputs. Here, we describe a series of assays to evaluate the effects of VEGF-A-stimulated intracellular signaling from intracellular compartments such as the endosome-lysosome system. These assays include the initial isolation and characterization of primary human endothelial cells, performing reverse genetics for analyzing protein function; methods used to study receptor trafficking, signaling, and proteolysis; and assays used to measure changes in cell migration, proliferation, and tubulogenesis. Each of these assays has been exemplified with studies performed in our laboratories. In conclusion, we describe necessary techniques for studying the role of VEGF-A in endothelial cell function.

Endosome-to-Plasma Membrane Recycling of VEGFR2 Receptor Tyrosine Kinase Regulates Endothelial Function and Blood Vessel Formation.

Rab GTPases are implicated in endosome-to-plasma membrane recycling, but how such membrane traffic regulators control vascular endothelial growth factor receptor 2 (VEGFR2/KDR) dynamics and function are not well understood. Here, we evaluated two different recycling Rab GTPases, Rab4a and Rab11a, in regulating endothelial VEGFR2 trafficking and signalling with implications for endothelial cell migration, proliferation and angiogenesis. In primary endothelial cells, VEGFR2 displays co-localisation with Rab4a, but not Rab11a GTPase, on early endosomes. Expression of a guanosine diphosphate (GDP)-bound Rab4a S22N mutant caused increased VEGFR2 accumulation in endosomes. TfR and VEGFR2 exhibited differences in endosome-to-plasma membrane recycling in the presence of chloroquine. Depletion of Rab4a, but not Rab11a, levels stimulated VEGF-A-dependent intracellular signalling. However, depletion of either Rab4a or Rab11a levels inhibited VEGF-A-stimulated endothelial cell migration. Interestingly, depletion of Rab4a levels stimulated VEGF-A-regulated endothelial cell proliferation. Rab4a and Rab11a were also both required for endothelial tubulogenesis. Evaluation of a transgenic zebrafish model showed that both Rab4 and Rab11a are functionally required for blood vessel formation and animal viability. Rab-dependent endosome-to-plasma membrane recycling of VEGFR2 is important for intracellular signalling, cell migration and proliferation during angiogenesis.

VEGF-A isoforms differentially regulate ATF-2-dependent VCAM-1 gene expression and endothelial-leukocyte interactions.

Vascular endothelial growth factor A (VEGF-A) regulates many aspects of vascular physiology. VEGF-A stimulates signal transduction pathways that modulate endothelial outputs such as cell migration, proliferation, tubulogenesis, and cell-cell interactions. Multiple VEGF-A isoforms exist, but the biological significance of this is unclear. Here we analyzed VEGF-A isoform-specific stimulation of VCAM-1 gene expression, which controls endothelial-leukocyte interactions, and show that this is dependent on both ERK1/2 and activating transcription factor-2 (ATF-2). VEGF-A isoforms showed differential ERK1/2 and p38 MAPK phosphorylation kinetics. A key feature of VEGF-A isoform-specific ERK1/2 activation and nuclear translocation was increased phosphorylation of ATF-2 on threonine residue 71 (T71). Using reverse genetics, we showed ATF-2 to be functionally required for VEGF-A-stimulated endothelial VCAM-1 gene expression. ATF-2 knockdown blocked VEGF-A-stimulated VCAM-1 expression and endothelial-leukocyte interactions. ATF-2 was also required for other endothelial cell outputs, such as cell migration and tubulogenesis. In contrast, VCAM-1 was essential only for promoting endothelial-leukocyte interactions. This work presents a new paradigm for understanding how soluble growth factor isoforms program complex cellular outputs and responses by modulating signal transduction pathways.