Bombesin, vasopressin, lysophosphatidic acid, and sphingosylphosphorylcholine induce focal adhesion kinase activation in intact Swiss 3T3 cells

Treatment of quiescent Swiss 3T3 cells with bombesin rapidly increased focal adhesion kinase (FAK)-associated tyrosine kinase activity in immune complexes. The effect was rapid (maximum at 2.5 min) and dose dependent (half-maximum response at 0.05 nM). Addition of vasopressin, lysophosphatidic acid, and sphingosylphosphorylcholine also elicited a rapid increase in FAK-associated tyrosine kinase activity. Addition of the selective Src inhibitor pyrazolopyrimidine directly to the in vitro kinase assay potently inhibited Src kinase activity induced by bombesin but did not affect the kinase activity of FAK measured by autophosphorylation or by synthetic substrate phosphorylation in paralell assays. In addition, Src activity was not detected in FAK immunoprecipitates using an optimal Src peptide substrate. Thus, agonist-induced tyrosine kinase activity measured in FAK immunoprecipitates is mediated by FAK activation rather than by co-immunoprecipitating Src. Bombesin-induced FAK activation is not dependent either on protein kinase C or Ca2+ mobilization but was completely blocked by treatment with cytochalasin D or by placing the cells in suspension. These findings indicate that FAK activation requires an intact actin cytoskeleton. Our results demonstrate that agonists that act via 7-transmembrane domain receptors stimulate FAK kinase activation.     

Phosphorylated cis-4-methylsphingosine mimics the mitogenic effect of sphingosine-1-phosphate in Swiss 3T3 fibroblasts

The phosphorylated derivative of sphingosine, sphingosine-1-phosphate, is a short-living metabolite of ultimate ceramide degradation and was shown to act as an intracellular signaling molecule, stimulating cell proliferation in quiescent Swiss 3T3 fibroblasts and inducing the release of calcium from intracellular stores (Zhang, H., Desai, N. N., Olivera, A., Seki, T., Brooker, G., and Spiegel, S. (1991) J. Cell. Biol. 114, 155-167). In the present study, 24-h treatment of Swiss 3T3 fibroblasts with the synthetic sphingosine analogue cis-4-methylsphingosine resulted in proliferation of quiescent Swiss 3T3 fibroblasts that was 3-fold stronger than that of equimolar sphingosine-1-phosphate. The phosphorylated derivative of cis-4-methylsphingosine accumulated drastically in the cells. Simultaneous treatment with the sphingosine kinase inhibitor L-threo-sphinganine reduced both the amount of phosphorylated cis-4-methylsphingosine and cell proliferation induced by this compound by about 50%, indicating that the phosphorylated derivative mediated the proliferative stimulus. The mitogenic effect of cis-4-methylsphingosine was associated with a mobilization of intracellular calcium in Swiss 3T3 fibroblasts that was similar to that induced by sphingosine-1-phosphate. The results demonstrate that the phosphorylated derivative of cis-4-methylsphingosine mimics the previously reported mitogenic action of sphingosine-1-phosphate in Swiss 3T3 cells, and the stronger effect most likely corresponds to the unusual accumulation of this compound.     

Cytotoxic necrotizing factor 1 from Escherichia coli and dermonecrotic toxin from Bordetella bronchiseptica induce p21(rho)-dependent tyrosine phosphorylation of focal adhesion kinase and paxillin in Swiss 3T3 cells

Treatment of Swiss 3T3 cells with cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli and dermonecrotic toxin (DNT) from Bordetella bronchiseptica, which directly target and activate p21(rho), stimulated tyrosine phosphorylation of focal adhesion kinase (p125(fak)) and paxillin. Tyrosine phosphorylation induced by CNF1 and DNT occurred after a pronounced lag period (2 h), and was blocked by either lysosomotrophic agents or incubation at 22 degrees C. CNF1 and DNT stimulated tyrosine phosphorylation of p125(fak) and paxillin, actin stress fiber formation, and focal adhesion assembly with similar kinetics. Cytochalasin D and high concentrations of platelet-derived growth factor disrupted the actin cytoskeleton and completely inhibited CNF1 and DNT induced tyrosine phosphorylation. Microinjection of Clostridium botulinum C3 exoenzyme which ADP-ribosylates and inactivates p21(rho) function, prevented tyrosine phosphorylation of focal adhesion proteins in response to either CNF1 or DNT. In addition, our results demonstrated that CNF1 and DNT do not induce protein kinase C activation, inositol phosphate formation, and Ca2+ mobilization. Moreover, CNF1 and DNT stimulated DNA synthesis without activation of p42(mapk) and p44(mapk) providing additional evidence for a novel p21(rho)-dependent signaling pathway that leads to entry into the S phase of the cell cycle in Swiss 3T3.     

A role for CAP, a novel, multifunctional Src homology 3 domain-containing protein in formation of actin stress fibers and focal adhesions

c-Cbl-associated protein, CAP, was originally cloned from a 3T3-L1 adipocyte cDNA expression library using full-length c-Cbl as a bait. CAP contains a unique structure, with three adjacent Src homology-3 (SH3) domains in the COOH terminus and a region sharing significant sequence similarity with the peptide hormone sorbin. Expression of CAP in NIH-3T3 cells overexpressing the insulin receptor induced the formation of stress fibers and focal adhesions. This effect of CAP expression on the organization of the actin-based cytoskeleton was independent of the type of integrin receptors engaged with extracellular matrix, whereas membrane ruffling and decreased actin stress fibers induced by insulin were not affected by expression of CAP. Immunofluorescence microscopy demonstrated that CAP colocalized with actin stress fibers. Moreover, CAP interacted with the focal adhesion kinase, p125FAK, both in vitro and in vivo through one of the SH3 domains of CAP. The increased formation of stress fibers and focal adhesions in CAP-expressing cells was correlated with decreased tyrosine phosphorylation of p125FAK in growing cells or upon integrin-mediated cell adhesion. These results suggest that CAP may mediate signals for the formation of stress fibers and focal adhesions.     

Involvement of tyrosine phosphorylation and protein kinase C in the activation of phospholipase D by H2O2 in Swiss 3T3 fibroblasts

We have investigated the mechanisms involved in H2O2-mediated phospholipase D (PLD) activation in Swiss 3T3 fibroblasts. In the presence of vanadate, H2O2 induced tyrosine phosphorylation of PLD as well as the platelet-derived growth (PDGF) factor receptor, protein kinase Calpha (PKCalpha), and a 62-kDa protein in rat brain PLD1 (rPLD1) immune complexes. PDGF also induced tyrosine phosphorylation of PLD, but this was abolished by catalase, indicating that it was mediated by H2O2 generation. Interestingly, PLD was found to be constitutively associated with the PDGF receptor and PKCalpha. Stimulation by H2O2 showed a concentration- and time-dependent tyrosine phosphorylation of the proteins in rPLD1 immunoprecipitates and activation of PLD in the cells. Pretreatment of the cells with the protein-tyrosine kinase inhibitors genistein and herbimycin A resulted in a concentration-dependent inhibition of H2O2-induced tyrosine phosphorylation and PLD activation. Activation of PLD by H2O2 was also inhibited dose-dependently by the PKC inhibitors Ro 31-8220 and calphostin C. Down-regulation of PKC by prolonged treatment with 4beta-phorbol 12-myristate 13-acetate also abolished H2O2-stimulated PLD activity. H2O2 or vanadate alone did not induce tyrosine phosphorylation of proteins in the rPLD1 immune complex or PLD activation. Reduction of intracellular H2O2 levels by pretreatment of the cells with catalase dramatically abrogated tyrosine phosphorylation of proteins in the rPLD1 immune complex and PLD activation, suggesting the potential role of intracellular H2O2 in H2O2-mediated PLD signaling. Taken together, these results suggest that both protein-tyrosine kinase(s) and protein kinase C participate in H2O2-induced PLD activation in Swiss 3T3 cells.     

Induction by interleukin-1beta peptide of prostaglandin E2 formation via enhanced prostaglandin H synthase-2 expression in 3T6 fibroblasts

Several synthetic interleukin-1 (IL-1) peptides were tested in vivo for pyrogenic activity and in vivo for their ability to stimulate prostaglandin production. Only the IL-1beta fragment (208-240) enhanced body temperature, although both IL-1beta (208-240) and IL-1alpha (223-250) stimulated prostaglandin E2 (PGE2) production in vitro. We report here that the IL-1beta fragment (208-240) did not have the capacity to induce arachidonic acid (AA) mobilization by 3T6 fibroblasts. However, this peptide was able to increase the expression of the inducible prostaglandin H synthase isoform (PGHS-2; EC 1.14.99.1.), which is related to its ability to stimulate prostaglandin E2 synthesis.     

CD4+ T lymphocytes migrating in three-dimensional collagen lattices lack focal adhesions and utilize beta1 integrin-independent strategies for polarization, interaction with collagen fibers and locomotion

Cell migration may depend on integrin-mediated adhesion to and deadhesion from extracellular matrix ligands. This concept, however, has not yet been confirmed for T lymphocytes migrating in three-dimensional extracellular matrices. We investigated receptor involvement in T cell migration combining a three-dimensional collagen matrix model with time-lapse videomicroscopy, computer-assisted cell tracking and confocal microscopy. In collagen lattices, the migration of CD4+ T cells (1) involved interactions with collagen fibers at the leading edge and uropod likewise, (2) occurred independently of the co-clustering of beta1, beta2, or beta3 integrins with F-actin, focal adhesion kinase, and phosphotyrosine at interactions with collagen fibers, (3) was counteracted by high-affinity beta1 integrin binding induced by antibody TS2/16; however, (4) the migration could not be blocked by a combination of adhesion-perturbing anti-beta1, -beta2, -beta3, and alpha v integrin antibodies. Integrin blocking neither affected cell polarization, interaction with fibers, beta1 integrin distribution, migration velocity, path structure, nor the number of locomoting cells in spontaneously migrating or concanavalin A-activated cells. Hence, T lymphocytes migrating in three-dimensional collagen matrices may utilize highly transient interactions with collagen fibers of low adhesivity, thereby differing from focal adhesion-dependent migration strategies employed by other cells.     

Activity of Rap1 is regulated by bombesin, cell adhesion, and cell density in NIH3T3 fibroblasts

Rap1 and Ras are homologous GTPases that are implicated in cell proliferation and differentiation. At present, little is known about the regulation of Rap1 activity. Using a recently developed assay with activation-specific probes, we found increased activity of endogenous Rap1 in NIH3T3 cells after stimulation with the neuropeptide growth factor bombesin in a concentration- and time-dependent manner. The activity of endogenous Ras was unaffected. Analysis of putative effectors showed no activation of c-Raf-1 or B-Raf after bombesin stimulation. However, MAPK/Erk-phosphorylation and the proliferation rate was increased. In addition, Rap1 was activated during cell adhesion to coated and uncoated tissue culture plates, as well as in response to various mitogens. Surprisingly, the basal Rap1 activity was observed to be cell density-dependent, with low levels when cells were reaching confluency. The results suggest that Rap1 acts as an important mediator of mitogenic signals distinct to Ras activation.     

Dissociation of mitogen-activated protein kinase activation from p125 focal adhesion kinase tyrosine phosphorylation in Swiss 3T3 cells stimulated by bombesin, lysophosphatidic acid, and platelet-derived growth factor

The experiments presented here were designed to examine the contribution of p125 focal adhesion kinase (p125FAK) tyrosine phosphorylation to the activation of the mitogen-activated protein kinase cascade induced by bombesin, lysophosphatidic acid (LPA), and platelet-derived growth factor (PDGF) in Swiss 3T3 cells. We found that tyrosine phosphorylation of p125FAK in response to these growth factors is completely abolished in cells treated with cytochalasin D or in cells that were suspended in serum-free medium for 30 min. In marked contrast, the activation of p42mapk by these factors was independent of the integrity of the actin cytoskeleton and of the interaction of the cells with the extracellular matrix. The protein kinase C inhibitor GF 109203X and down-regulation of protein kinase C by prolonged pretreatment of cells with phorbol esters blocked bombesin-stimulated activation of p42mapk, p90rsk, and MAPK kinase-1 but did not prevent bombesin-induced tyrosine phosphorylation of p125FAK. Furthermore, LPA-induced p42mapk activation involved a pertussis toxin-sensitive guanylate nucleotide-binding protein, whereas tyrosine phosphorylation of p125FAK in response to LPA was not prevented by pretreatment with pertussis toxin. Finally, PDGF induced maximum p42mapk activation at concentrations (30 ng/ml) that failed to induce tyrosine phosphorylation of p125FAK. Thus, our results demonstrate that p42mapk activation in response to bombesin, LPA, and PDGF can be dissociated from p125FAK tyrosine phosphorylation in Swiss 3T3 cells.     

Role of mitogen-activated protein kinases and c-Jun/AP-1 trans-activating activity in the regulation of protease mRNAs and the malignant phenotype in NIH 3T3 fibroblasts

Ras activates a multitude of downstream activities with roles in cellular proliferation, invasion and metastasis, differentiation, and programmed cell death. In this work we have evaluated the requirement of extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal kinase kinase (JNKK), and c-Jun/AP-1 activities in transformation and extracellular matrix invasion of ras oncogene expressing NIH 3T3 fibroblasts by expressing stable mutant genes that constitutively inhibit these activities. Whereas the inhibition of ERK activity reverts the transformed and invasive phenotype, the inhibition of the JNK pathway and AP-1 trans-activating activities by JNKK[K129R] and c-Jun(TAM67) had no effect on the ability of the ras oncogene-expressing cells to grow in soft agar or invade Matrigel basement membrane. Thus an elevated JNK activity and/or c-Jun/AP-1 trans-activating activity are not absolute requirements for ras transformation or invasion through basement membrane, and the dependence on AP-1 activity for transformation is cell-specific. However, inhibition of JNK kinase (JNKK) in ras-transformed cells with normally elevated JNK activity switches the protease-dependent invasive phenotype from a urokinase plasminogen activator (uPA)-dependent to a cathepsin L (CL)-dependent invasive phenotype. Conversely, treatment of ras-transformed cells of low constitutive JNK activity with the JNK stimulator, anisomycin, converts the protease mRNA levels from those characteristic of a CL-dependent to a uPA-dependent phenotype. These protease phenotypes can be duplicated in untransformed NIH 3T3 cells that express platelet-derived growth factor receptors and m1 muscarinic receptors that selectively stimulate the ERK or JNK pathways, respectively. It is concluded that high ERK activity is required for both protease phenotypes, whereas the JNK pathway and c-Jun/AP-1 activity are not required for transformation but regulate a switch between uPA and CL protease phenotypes in both transformed and untransformed cells. In ras-transformed NIH 3T3 fibroblasts, the uPA- and CL-dependent protease phenotypes are redundant in their ability to invade through basement membrane.     

T cell receptor- and beta 1 integrin-mediated signals synergize to induce tyrosine phosphorylation of focal adhesion kinase (pp125FAK) in human T cells

The beta 1 subfamily of integrins is thought to play an important role in both the adhesion/migration and proliferation/differentiation of T cells. beta 1 integrins can provide T cell costimulation through interaction of very late antigen (VLA) 4 (VLA-4) (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) with the extracellular matrix protein fibronectin (FN), or by VLA-4 binding to its cell surface ligand, vascular cell adhesion molecule (VCAM) 1. The mechanism by which beta 1 integrin members transduce T cell-costimulatory signals is poorly understood. Studies in non-T cells have demonstrated regulation of the tyrosine focal adhesion kinase pp125FAK by beta 1 integrin engagement and, most recently, indicate a role for pp125FAK in linking integrin-mediated signal transduction to the Ras pathway (Schaller, M. D., and J. T. Parsons, 1994, Curr. Opin. Cell. Biol. 6: 705-710; Schlaepfer, D. D., S. K. Hanks, T. Hunter, and P. Van der Geer. 1994. Nature (Lond.), 372:786-790). Although pp125FAK kinase occurs in T cells, there are no reports on its regulation in this cell type. The studies described in this article characterize novel regulation of pp125FAK by the T cell receptor (TCR)-CD3 antigen complex and beta 1 integrins, and provide the first account, in any cell type, of integrin alpha 4 beta 1-mediated pp125FAK tyrosine phosphorylation. We demonstrate a rapid and sustained synergistic increase in tyrosine phosphorylation of human pp125FAK in Jurkat T cells after simultaneous (a) triggering of the TCR-CD3 complex, and (b) alpha 4 beta 1 and alpha 5 beta 1 integrin-mediated binding of these cells to immobilized FN or alpha 4 beta 1 integrin-mediated binding to immobilized VCAM-1. Studies with normal peripheral blood-derived CD4+ human T blasts confirm the synergistic action of a TCR-CD3 complex-mediated costimulus with a FN- or VCAM-1-dependent signal in the induction of T cell pp125FAK tyrosine phosphorylation. In vitro kinase assays performed on pp125FAK immunoprecipitates isolated from Jurkat cells and normal CD4+ T cells identified a coprecipitating 57-kD tyrosine-phosphorylated protein (pp57), distinct from pp59fyn or pp56lck. These results indicate, for the first time, the involvement of a specific kinase, pp125FAK, in alpha 4 beta 1- and alpha 5 beta 1-mediated T cell-costimulatory signaling pathways. In addition, the data demonstrate novel regulation of pp125FAK tyrosine phosphorylation by the TCR-CD3 complex.     

flt3 ligand in cooperation with transforming growth factor-beta1 potentiates in vitro development of Langerhans-type dendritic cells and allows single-cell dendritic cell cluster formation under serum-free conditions

Using a recently described serum-free culture system of purified human CD34+ progenitor cells, we show here a critical cooperation of flt3 ligand (FL) with transforming growth factor-beta1 (TGF-beta1) in the induction of in vitro dendritic cell/Langerhans cell (DC/LC) development. The addition of FL to serum-free cultures of CD34+ cells supplemented with TGF-beta1, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor alpha, and stem cell factor strongly increases both percentages (mean, 36% +/- 5% v 64% +/- 4%; P = .001) and total numbers (4.4- +/- 0.8-fold) of CD1a+ dendritic cells. These in vitro-generated CD1a+ cells molecularly closely resemble a particular type of DC known as an epidermal Langerhans cell. Generation of DC under serum-free conditions was found to strictly require supplementation of culture medium with TGF-beta1. Upon omission of TGF-beta1, percentages of CD1a+ DC decreased (to mean, 10% +/- 8%; P = .001) and, in turn, percentages of granulomonocytic cells (CD1a- cells that are lysozyme [LZ+]; myeloperoxidase [MPO+]; CD14+) increased approximately threefold (P < .05). Furthermore, in the absence of TGF-beta1, FL consistently promotes generation of LZ+, MPO+, and CD14+ cells, but not of CD1a+ cells. Serum-free single-cell cultures set up under identical TGF-beta1- and FL-supplemented culture conditions showed that high percentages of CD34+ cells (mean, 18% +/- 2%; n = 4) give rise to day-10 DC colony formation. The majority of cells in these DC-containing colonies expressed the Langerhans cell/Birbeck granule specific marker molecule Lag. Without TGF-beta1 supplementation, Lag+ colony formation is minimal and formation of monocyte/macrophage-containing colonies predominates. Total cloning efficiency in the absence and presence of TGF-beta1 is virtually identical (mean, 41% +/- 6% v 41% +/- 4%). Thus, FL has the potential to strongly stimulate DC/LC generation, but has a strict requirement for TGF-beta1 to show this costimulatory effect.     

Sodium butyrate induces NIH3T3 cells to senescence-like state and enhances promoter activity of p21WAF/CIP1 in p53-independent manner

PURPOSE: To evaluate the diagnostic efficacy of computed tomography (CT) after hepatic intraarterial injection of iodized oil in patients with hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Forty patients who underwent CT with iodized oil before orthotopic liver transplantation (OLT) were evaluated prospectively. All patients underwent digital subtraction angiography and injection of iodized oil during chemoembolization. CT during arterial portography (CTAP) was performed in 34 patients. The number of neoplastic nodules was assessed in explanted livers and compared with the radiologic results. RESULTS: Sixty-six HCC nodules were present in the explanted livers. CT with iodized oil enabled correct diagnosis in 38 of 66 lesions (58%), and the results were false-positive in two lesions (3%). Digital subtraction angiography had a sensitivity of 67% (44 of 66 nodules) and CTAP had a sensitivity of 85% (45 of 53 nodules). Four (6%) false-positive diagnoses were made at digital subtraction angiography and three (6%) at CTAP. The diagnostic efficacy of CT with iodized oil was significantly related to lesion diameter greater than 2 cm (P < .0001) and hypervascularity (P < .0001). CONCLUSION: CT with iodized oil failed to provide any substantial information in the pre-OLT staging of HCC: It was inaccurate for small HCC nodules (<2 cm) and intrahepatic metastases. Its sensitivity matched that of digital subtraction angiography and was statistically significantly inferior to that of CTAP.     

HLA-independent heterogeneity of CD8+ T cell responses to MAGE-3, Melan-A/MART-1, gp100, tyrosinase, MC1R, and TRP-2 in vaccine-treated melanoma patients

An important element in melanoma vaccine construction is to identify peptides from melanoma-associated Ags that have immunogenic potential in humans and are recognized by CD8+ T cells in vivo. To identify such peptides, we evaluated HLA-A*02+ melanoma patients immunized to a polyvalent vaccine containing multiple Ags, including MAGE-3, Melan-A/MART-1, gp100, tyrosinase, melanocortin receptor (MC1R), and dopachrome tautomerase (TRP-2). Using a filter spot assay, we measured peripheral blood CD8+ T cell responses, before and after immunization, to a panel of 45 HLA-A*0201-restricted peptides derived from these Ags. The peptides were selected for immunogenic potential based on their strong binding affinity in vitro to HLA-A*0201. Vaccine treatment induced peptide-specific CD8+ T cell responses to 22 (47.8%) of the peptides. The most striking finding was the HLA-independent heterogeneity of responses to both peptides and Ags. All responding patients reacted to different combination of peptides and Ags even though the responding patients were all A*0201+ and the peptides were all A*0201-restricted. From 9 to 27% of patients developed a CD8+ T cell response to at least one peptide from each Ag, but no more than 3 (14%) reacted to the same peptide from the same Ag. This heterogeneity of responses to individual peptides and Ags in patients with the same haplotype points to the need to construct vaccines of multiple peptides or Ags to maximize the proportion of responding patients.