R. L. Juliano

Integrin signaling and cell growth control

Integrins contribute to cell growth by providing a physical linkage between cytoskeletal structures and the extracellular matrix, and also by participating in various signal transduction processes. The interaction of integrins with matrix ligands can generate signals in and of itself, and can also modulate signals instigated by soluble factors such as peptide mitogens. Cellular events affected by integrin-mediated signaling include motility, cell division, differentiation and programmed cell death. Elucidation of how integrin-mediated cell adhesion controls cell growth is likely to be of fundamental importance in understanding complex biological processes, such as tissue morphogenesis and tumor progression.

Anchorage-dependent ERK signaling - Mechanisms and consequences

Integrin-mediated adhesion to the extracellular matrix regulates the cellular response to mitogens. Anchorage-dependent growth factor activation of the extracellular signal-regulated kinase (ERK) is an intensely studied example of this regulation. Given the central role of ERK in mediating cell migration, division, and survival, it is also an extremely important example. Recent work has demonstrated that cell adhesion can regulate ERK signaling at several checkpoints and has begun to define the mechanism and consequences associated with anchorage-dependent effects on the ERK cascade.

Integrin regulation of cell signalling and motility

Integrins clearly play a key role in regulating both mitogenic signalling and cell migration. Thus integrins modulate the efficiency of the Erk (extracellular-signal-regulated kinase)/MAP kinase (mitogen-activated protein kinase) pathway, acting at several distinct levels. We have shown that both cAMP-dependent protein kinase and PAKs (p21-activated kinases) play a role in integrin regulation of the Erk pathway, acting primarily at the level of Raf-1. Integrins and PAKs also play a role in the control of cell migration. Thus we have discovered a novel protein that links the alpha5beta1 integrin to migration controlled by Rho-family GTPases. This protein, termed Nischarin, is a large cytosolic macromolecule that is not related to well-known protein families. The N-terminus of Nischarin interacts with a short segment of the cytoplasmic domain of the alpha5 integrin subunit. Overexpression of Nischarin alters actin organization and inhibits Rac-driven cell migration and tumour cell invasion. Use of effector domain mutants of Rac suggest that Nischarin acts downstream of Rac, probably at the level of PAK-family kinases. These studies emphasize the intricate connection between integrins and Rho-family GTPases and their effectors in controlling both mitogenesis and migration.

Biological aspects of signal transduction by cell adhesion receptors

Cell adhesion receptors such as integrins, cadherins, selectins, and immunoglobulin family receptors profoundly modulate many signal transduction cascades. In this review we examine aspects of adhesion receptor signaling and how this impinges on key biological processes. We have chosen to focus on cell migration and on programmed cell death. We examine many of the cytoplasmic signaling molecules that interface with adhesion receptors, including focal adhesion kinase (FAK), phosphatidylinositol-3-kinase (PI3K), and elements of the Erk/MAP kinase pathway. In many cases these molecules impinge on both the regulation of cell movement and on control of apoptosis.

Regulation of nucleocytoplasmic trafficking by cell adhesion receptors and the cytoskeleton

It has become widely accepted that adhesion receptors can either directly activate, or significantly modulate, many of the signaling cascades initiated by circulating growth factors. An interesting recent development is the realization that adhesion receptors and their cytoskeletal partners can regulate the trafficking of signaling proteins between the cytoplasm and nucleus. Cell adhesion molecule control of nucleocytoplasmic trafficking allows adhesion to influence many cell decisions, and highlights the diversity of nuclear import and export mechanisms.

Anchorage-dependent Regulation of the Mitogen-activated Protein Kinase Cascade by Growth Factors Is Supported by a Variety of Integrin α Chains

Integrin cooperation with growth factor receptors to enable permissive signaling to the mitogen-activated protein (MAP) kinase pathway has important implications for cell proliferation, differentiation, and survival. Here we have sought to determine whether anchorage regulation of the MAP kinase pathway is specific to the α chain subunit of the integrins employed during adhesion. Human umbilical vein endothelial cells (HUVECs) anchored via endogenous α2, α3, or α5 integrin subunits or NIH3T3 fibroblast cells lines anchored via ectopically expressed human integrin α2 or α5 subunits displayed comparable MAP kinase activation upon growth factor stimulation, regardless of the integrin α chain employed. In contrast, when either cell type was maintained in suspension, growth factor treatment inefficiently activated the MAP kinase pathway. The integrin-mediated enhancement of MAP kinase activation by growth factor correlated with the tyrosine phosphorylation of focal adhesion kinase but was independent of Shc. These data indicate that integrin modulation of the MAP kinase pathway is supported by a variety of integrin complexes and imply that other pathways may be required for the previously reported α chain-specific effects on cell cycle regulation and cell differentiation.

Evaluation of strategies for the intracellular delivery of proteins

AbstractPurpose. The intracellular delivery of functionally active proteins represents an important emerging strategy for laboratory investigation and therapeutic applications. Although a number of promising approaches for protein delivery have been developed, thus far there has been no attempt to compare the merits of the various delivery technologies. This issue is addressed in the current study.nMethods. In this study we utilize a sensitive luciferase reporter gene assay to provide unambiguous and quantitative evaluation of several strategies for the intracellular delivery of a biologically active protein comprised of the Gal4 DNA binding domain and the VP16 transactivating domain.nResults. Both a cationic lipid supramolecular complex and a polymeric complex were able to effectively deliver the chimeric transcription factor to cultured cells. In addition, protein chimeras containing the Tat cell penetrating peptide, but not those containing the VP22 peptide, were somewhat effective in delivery.nConclusions. Both supramolecular protein-carrier complexes and protein chimeras with certain cell penetrating peptides can support intracellular delivery of proteins. In the cell culture setting the supramolecular complexes are more effective, but their large size may present problems for in vivo applications.

Challenges to macromolecular drug delivery

The use of macromolecules, particularly monoclonal antibodies, as therapeutic agents has come to the forefront in recent years. The biodistribution and delivery issues for protein drugs are shared to a substantial degree with other emerging therapeutic approaches including pharmacologically active nucleic acids and nanoparticles. A generalized approach to these issues involves consideration of the multiple biological barriers that stand between the macromolecular drug or nanoparticle at its site of administration and its ultimate biological target. Considerations of size, stability, non-specific versus specific associations and potency versus toxicity all play a role. The creation of delivery approaches that combine high specificity for the target cell or tissue, high therapeutic payload and modest toxicity remains a challenge, although some very promising examples have emerged recently. A variety of sophisticated targeting strategies, based primarily on combinatorial library methods, when used in combination with new technologies to identify cell-surface receptor signatures of specific tissues, will facilitate advances in targeted delivery of macromolecules and nanoparticles. The challenges to contemporary macromolecule drug delivery are complex, thus new research paradigms are emerging that combine the talents of physical and biological scientists to address this key issue for modern pharmacology and therapeutics.

IN VITRO TRANSPORT AND DELIVERY OF ANTISENSE OLIGONUCLEOTIDES

A variety of techniques are currently available to enhance the cellular uptake and pharmacological effectiveness of antisense oligonucleotides in the in vitro setting. The choice of technique will depend on the context of investigation, the likelihood of cytotoxity due to the delivery agents, and the ease and convenience of the approach. The considerations for the delivery of antisense molecules in the in vivo setting are likely to be quite different from the cell culture situation emphasized in this article.

Integrin regulation of receptor tyrosine kinase and G protein-coupled receptor signaling to mitogen-activated protein kinases.

Assays for use in integrin-mediated or -modulated signaling are essentially the same as those used in signaling studies involving growth factors or hormones. The major differences are the manipulations of the cells to compare the effects of gain or loss of anchorage, or the role of specific adhesion receptors. This needs to be done with some care, and with thought given to the overall biology of the particular cell type under investigation. Harsh treatment of cells, for example, prolonged suspension culture, may result in irreversible nonphysiological effects in some types of cells.