Alterations in the focal adhesion kinase/Src signal transduction pathway correlate with increased migratory capacity of prostate carcinoma cells.

Focal adhesion kinase (FAK) has been implicated in the regulation of cell migration. In addition, FAK expression is increased in a number of highly metastatic tumor cell lines. Therefore, we investigated the role of FAK in regulating migration of prostate carcinoma cell lines with increasing metastatic potential. We show that highly tumorigenic PC3 and DU145 cells exhibit intrinsic migratory capacity, while poorly tumorigenic LNCaP cells require a stimulus to migrate. Increased metastatic potential of PC3 and DU145 cells correlates with increased FAK expression, overall tyrosine phosphorylation and activity, as measured by autophosphorylation of tyrosine 397. However, in PC3 and DU145 cells, FAK autophosphorylation is adhesion dependent whereas a second site of tyrosine phosphorylation, tyrosine 861, a Src specific site, is uncoupled from adhesion-dependent signaling events. Finally, inhibiting the FAK/Src signal transduction pathway by over expressing FRNK (Focal adhesion kinase-Related Non-Kinase), an inhibitor of FAK activation, or treatment with PP2, a Src family kinase inhibitor, significantly inhibited migration of prostate carcinoma cell lines, demonstrating that tumor cell migration continues to be dependent on signals emanating from this pathway.

Focal adhesion kinase: a regulator of focal adhesion dynamics and cell movement.

Engagement of integrin receptors with extracellular ligands gives rise to the formation of complex multiprotein structures that link the ECM to the cytoplasmic actin cytoskeleton. These adhesive complexes are dynamic, often heterogeneous structures, varying in size and organization. In motile cells, sites of adhesion within filopodia and lamellipodia are relatively small and transient and are referred to as 'focal complexes,' whereas adhesions underlying the body of the cell and localized to the ends of actin stress fibers are referred to as 'focal adhesions'. Signal transduction through focal complexes and focal adhesions has been implicated in the regulation of a number of key cellular processes, including growth factor induced mitogenic signals, cell survival and cell locomotion. The formation and remodeling of focal contacts is a dynamic process under the regulation of protein tyrosine kinases and small GTPases of the Rho family. In this review, we consider the role of the focal complex associated protein tyrosine kinase, Focal Adhesion Kinase (FAK), in the regulation of cell movement with the emphasis on how FAK regulates the flow of signals from the ECM to the actin cytoskeleton.

Attenuation of plasminogen activator inhibitor type-1 promoter activity in serum-stimulated renal epithelial cells by a distal 5' flanking region.

Urokinase plasminogen activator (u-PA) and its fast acting type-1 inhibitor (PAI-1) localize to cellular focal adhesive structures and the adjoining proximal undersurface region, respectively (Kutz et al., J. Cell. Physiol. 176:8-18, 1997). PAI-1 may function in this locale to modulate pericellular proteolytic activity, cell-to-substrate adhesion, or matrix-dependent motility. While PAI-1 synthesis is regulated in an immediate-early response manner in growth "activated" renal cells coincident with cytoskeletal restructuring, adhesive influences both repress the amplitude and prolong the time course of serum-induced PAI-1 transcription (Ryan et al., Biochem. J. 314:1041-1046, 1996). To identify potential adhesion-responsive elements within the PAI-1 gene that function in this complex mode of expression control, reporter constructs containing defined directionally deleted PAI-1 5' genomic fragments cloned upstream of a CAT gene were employed in transient transfection assays. A 483-bp distal PAI-1 flanking segment (corresponding to nucleotides -2395 to -1912) conferred significant adhesion-dependent attenuation on serum-induced PAI-1 transcription. This 483-bp distal PAI-1 segment functioned as a repressor of reporter (CAT) activity under both adhesive and suspension culture conditions, however, when ligated upstream of either an SV40 promoter/enhancer or a minimal PAI-1 promoter. These data suggest that repressor elements located between -2395 and -1912 bp interact with more proximal adhesion-dependent regulatory elements to affect PAI-1 expression attenuation during serum stimulation of adherent renal epithelial cells.

c-Raf-mediated inhibition of epidermal growth factor-stimulated cell migration.

Epidermal growth factor stimulates migration of a number of cell types, yet the signaling pathways that regulate epidermal growth factor-stimulated migration are poorly defined. In this report, we employ a transient transfection migration assay to assess the role of components of the Ras-mitogen-activated protein (MAP) kinase signaling pathway in epidermal growth factor-stimulated chemotaxis of rat embryo fibroblasts. Expression of dominant negative Ras blocks epidermal growth factor-mediated chemotaxis, while constitutively active Ras has no effect on chemokinesis or chemotaxis. PD98059 and U0126, inhibitors of MAP kinase kinase (MEK) activity, decreased epidermal growth factor-stimulated migration, while kinase-defective MEK1, an inhibitor of MAP kinase activation, enhanced migration. To understand the paradoxical effects of these molecules on epidermal growth factor-induced migration, we examined the role of c-Raf on migration. Expression of either wild type c-Raf or the catalytic domain of c-Raf effectively inhibited epidermal growth factor-stimulated cell migration. We suggest that, whereas Ras activity is necessary to promote epidermal growth factor-stimulated migration, sustained activation of c-Raf may be important in down-regulating migratory signaling pathways triggered by epidermal growth factor receptor activation. Further, activation of c-Raf upon inhibition of the MEK-MAP kinase pathway may contribute to the inhibition of cell migration observed with pharmacological MEK inhibitors.

Differential regulation of PAI-1 gene expression in human fibroblasts predisposed to a fibrotic phenotype.

Synthesis of the major negative physiologic regulator of plasmin activation [plasminogen activator inhibitor type-1 (PAI-1)] is elevated during progressive cellular senescence, in premature aging disorders (e.g., Werner's syndrome), and in conditions associated with tissue fibrosis and excessive fibrin accumulation (e.g., sclerosis, keloid formation). Dermal fibroblasts derived from Werner's patients as well as from keloid lesions markedly overexpress PAI-1 mRNA transcripts compared to normal skin fibroblasts. Such cell type-related differences in steady-state PAI-1 mRNA content, and variances in the relative abundance of the 3.0- compared to the 2.2-kb PAI-1 mRNA species, served to discriminate normal from Werner's and keloid fibroblasts. This disparity in PAI-1 mRNA levels paralleled transcriptional activities of the PAI-1 gene; de novo synthesis of PAI-1 protein among the three cell types, moreover, closely approximated the respective differences in total PAI-1 mRNA content. Despite the markedly elevated levels of PAI-1 mRNA and protein evident in newly confluent keloid fibroblasts, these cells effectively suppressed PAI-1 synthesis (as did normal dermal fibroblasts) upon culture in serum-free medium. Werner's syndrome skin fibroblasts, in contrast, continued to maintain high-level PAI-1 expression even after 3 days of growth arrest. Adhesion-mediated attenuation of serum-stimulated PAI-1 expression, a characteristic of normal cells involving sequences which mapped to the distal 5' flanking region of the PAI-1 gene, was retained in keloid but not Werner's fibroblasts. Collectively, these data suggest that (1) specific controls on PAI-1 gene expression are fundamentally different between these two clinically significant high PAI-1-synthesizing cell types and (2) the localized keloid may define the emergence of a distinct profibrotic dermal fibroblastoid phenotype in genetically predisposed individuals.

Cytoarchitecture and cell growth control.

Appropriate cell-to-substrate adhesion together with SGF stimulation is necessary to initiate and continue cell cycle progression of growth arrested cells. Adhesion-dependent signaling events, which likely occur through integrin receptors specifically organized with cytoskeletal components within focal contacts, can induce expression of specific genes and stimulate quiescent cells into the growth cycle. The mechanisms as to how: (1) cell-to-substrate adhesion complexes are formed and maintained, (2) adhesion-dependent signal transduction events interface with SGF initiated signalling events, (3) adhesion influences expression of growth-state regulated genes, and (4) an appropriate cytoarchitectural environment may coordinate these events to regulate cellular growth are unclear. While it is apparent that defining these mechanisms would be critical to understanding the basic events which control cell growth, many of the mechanisms are just beginning to be addressed and understood.

Sigmoid neural transfer function realized by percolation.

An experiment using the phenomenon of percolation has been conducted to demonstrate the implementation of neural functionality (summing and sigmoid transfer). A simple analog approximation to digital percolation is implemented. The device consists of a piece of amorphous silicon with stochastic bit-stream optical inputs, in which a current percolating from one end to the other defines the neuron output, also in the form of a stochastic bit stream. Preliminary experimental results are presented.

Biased T-cell receptor delta element recombination in scid thymocytes.

Thymocytes in mutant mice with severe combined immunodeficiency (scid thymocytes) show ongoing recombination of some T-cell receptor delta gene elements, generating signal joints quantitatively and qualitatively indistinguishable from those in wild-type fetal thymocytes. Excised D delta 2-J delta 1 and D delta 1-D delta 2 rearrangements are detectable at levels equivalent to or greater than those in thymocytes from wild-type mice on fetal day 15. Signal junctional modification, shown here to occur frequently in wild-type adult but not newborn excised D delta 2-J delta 1 junctions, can occur normally in adult scid thymocytes. Excised D delta 1-D delta 2 scid junctions, similar to wild-type thymocytes, include pseudonormal coding junctions as well as signal junctions. Inversional D delta 1-D delta 2 rearrangements, generating conventional hybrid junctions, are also reproducibly detectable in scid thymus DNA. These hybrids, unlike those reported for artificial recombination constructs, do not show extensive nucleotide loss. In contrast to the normal or high incidences of D delta 1-, D delta 2-, and J delta 1-associated signal junctions in scid thymocytes, V delta 1, V gamma 3, and V gamma 1.2 signal products are undetectable in scid thymocytes or are detectable at levels at least 10-fold lower than the levels in wild-type fetal thymocytes. These findings confirm biased T-cell receptor element recombination by V(D)J recombinase activity of nontransformed scid thymocytes and indicate that analysis of in vivo-mediated gene rearrangements is important for full understanding of how the scid mutation arrests lymphocyte development.

V(D)J recombination generates a high frequency of nonstandard TCR D delta-associated rearrangements in thymocytes.

The standard products of V(D)J recombination are coding junctions, which encode Ag receptor polypeptides, and their commonly excised reciprocal products, signal junctions. Additional nonstandard products also have been detected, mostly in artificial recombination substrate studies. The occurrence of nonstandard products, including pseudonormal, hybrid, and open/shut junctions, indicates significant indeterminacy of the V(D)J recombinase. However, the incidence of nonstandard products for endogenous Ag receptor genes in vivo has not been specifically addressed. The data presented here show that for the TCR-delta locus, D element-associated recombination in mouse thymocytes results in a high incidence of nonstandard recombination products. D delta 1-D delta 2 rearrangements, both chromosome retained and excised episomal products, were studied by polymerase chain reaction amplification, cloning, and sequence analysis. The proximity of D delta 1 and D delta 2 elements, and the fact that both are flanked by 5' and 3' recombination signal sequences with 12-bp and 23-bp spacers, respectively, results in frequent pseudonormal joining. The resulting products are signal junctions retained on the chromosome. Excised episomal products include coding junctions, hybrid junctions formed in apparent violation of the 12/23 spacer rule, and standard signal junctions; some signal junctions show evidence of imprecise cleavage. Evidence for open/shut and/or oligonucleotide capture events was also seen. Similar rearrangements were detectable in thymocytes of mutant scid mice. These findings indicate a high degree of indeterminacy of V(D)J recombinase-mediated D delta 1-D delta 2 rearrangement in both wild-type and scid thymocytes. This indeterminacy affects the productive potential of TCR-delta loci.