Mark R. Morgan

Synergistic control of cell adhesion by integrins and syndecans.

The ability of cells to adhere to each other and to their surrounding extracellular matrices is essential for a multicellular existence. Adhesion provides physical support for cells, regulates cell positioning and enables microenvironmental sensing. The integrins and the syndecans are two adhesion receptor families that mediate adhesion, but their relative and functional contributions to cell–extracellular matrix interactions remain obscure. Recent advances have highlighted connections between the signalling networks that are controlled by these families of receptors. Here we survey the evidence that synergistic signalling is involved in controlling adhesive function and the regulation of cell behaviour in response to the external environment.

Syndecan-4-dependent Rac1 regulation determines directional migration in response to the extracellular matrix.

Cell migration in wound healing and disease is critically dependent on integration with the extracellular matrix, but the receptors that couple matrix topography to migratory behavior remain obscure. Using nano-engineered fibronectin surfaces and cell-derived matrices, we identify syndecan-4 as a key signaling receptor determining directional migration. In wild-type fibroblasts, syndecan-4 mediates the matrix-induced protein kinase Cα (PKCα)–dependent activation of Rac1 and localizes Rac1 activity and membrane protrusion to the leading edge of the cell, resulting in persistent migration. In contrast, syndecan-4–null fibroblasts migrate randomly as a result of high delocalized Rac1 activity, whereas cells expressing a syndecan-4 cytodomain mutant deficient in PKCα regulation fail to localize active Rac1 to points of matrix engagement and consequently fail to recognize and respond to topographical changes in the matrix.

p190RhoGAP is the convergence point of adhesion signals from alpha 5 beta 1 integrin and syndecan-4.

The fibronectin receptors α5β1 integrin and syndecan-4 cocluster in focal adhesions and coordinate cell migration by making individual contributions to the suppression of RhoA activity during matrix engagement. p190Rho–guanosine triphosphatase–activating protein (GAP) is known to inhibit RhoA during the early stages of cell spreading in an Src-dependent manner. This paper dissects the mechanisms of p190RhoGAP regulation and distinguishes the contributions of α5β1 integrin and syndecan-4. Matrix-induced tyrosine phosphorylation of p190RhoGAP is stimulated solely by engagement of α5β1 integrin and is independent of syndecan-4. Parallel engagement of syndecan-4 causes redistribution of the tyrosine-phosphorylated pool of p190RhoGAP between membrane and cytosolic fractions by a mechanism that requires direct activation of protein kinase C α by syndecan-4. Activation of both pathways is necessary for the efficient regulation of RhoA and, as a consequence, focal adhesion formation. Accordingly, we identify p190RhoGAP as the convergence point for adhesive signals mediated by α5β1 integrin and syndecan-4. This molecular mechanism explains the cooperation between extracellular matrix receptors during cell adhesion.

A syndecan-4 hair trigger initiates wound healing through caveolin- and RhoG-regulated integrin endocytosis.

Summary Cell migration during wound healing requires adhesion receptor turnover to enable the formation and disassembly of cell-extracellular matrix contacts. Although recent advances have improved our understanding of integrin trafficking pathways, it is not known how extracellular ligand engagement controls receptor dynamics. Using atomic force microscopy, we have measured cell avidity for fibronectin and defined a mechanism for the outside-in regulation of α5β1-integrin. Surprisingly, adhesive strength was attenuated by the syndecan-4-binding domain of fibronectin due to a rapid triggering of α5β1-integrin endocytosis. Association of syndecan-4 with PKCα was found to trigger RhoG activation and subsequent dynamin- and caveolin-dependent integrin uptake. Like disruption of syndecan-4 or caveolin, gene disruption of RhoG in mice was found to retard closure of dermal wounds due to a migration defect of the fibroblasts and keratinocytes of RhoG null mice. Thus, this syndecan-4-regulated integrin endocytic pathway appears to play a key role in tissue repair.

Syndecan-4 Phosphorylation Is a Control Point for Integrin Recycling

Summary Precise spatiotemporal coordination of integrin adhesion complex dynamics is essential for efficient cell migration. For cells adherent to fibronectin, differential engagement of α5β1 and αVβ3 integrins is used to elicit changes in adhesion complex stability, mechanosensation, matrix assembly, and migration, but the mechanisms responsible for receptor regulation have remained largely obscure. We identify phosphorylation of the membrane-intercalated proteoglycan syndecan-4 as an essential switch controlling integrin recycling. Src phosphorylates syndecan-4 and, by driving syntenin binding, leads to suppression of Arf6 activity and recycling of αVβ3 to the plasma membrane at the expense of α5β1. The resultant elevation in αVβ3 engagement promotes stabilization of focal adhesions. Conversely, abrogation of syndecan-4 phosphorylation drives surface expression of α5β1, destabilizes adhesion complexes, and disrupts cell migration. These data identify the dynamic spatiotemporal regulation of Src-mediated syndecan-4 phosphorylation as an essential switch controlling integrin trafficking and adhesion dynamics to promote efficient cell migration.

Giving off mixed signals—Distinct functions of α5β1 and αvβ3 integrins in regulating cell behaviour

The formation, maturation, and dissolution of focal adhesions are basic prerequisites of cell migration and rely on the recruitment, signalling, and endocytosis of integrins. In many instances, extracellular matrix molecules are recognised by a number of integrins, and it is the sequential involvement of different integrins that allows establishment of cell polarity and migration towards a matrix stimulus. In this review, we consider both the similarities and differences between two key fibronectin receptors, αvβ3 and α5β1 integrin. By considering the GTPase and kinase signalling and trafficking of two such closely‐related receptors, we begin to understand how cell migration is coordinated.

Syndecans shed their reputation as inert molecules

The shedding of the extracellular domain of a transmembrane proteoglycan can be controlled by its cytoplasmic domain. The syndecan transmembrane proteoglycans synergize with receptors for extracellular matrix molecules and growth factors to initiate cytoplasmic signals in response to a range of extracellular stimuli. Syndecans influence a wide range of physiological processes, but their contribution is most apparent during wound repair. Aspects of syndecan biology that have attracted research interest include extracellular matrix binding, outside-to-inside plasma membrane signal propagation, activation of cytoplasmic signals, and shedding of the syndecan extracellular domain, but the mechanisms by which syndecan cytoplasmic signals modulate extracellular function remain largely unresolved. Hayashida et al. have now discovered that association between an endocytic regulator, Rab5, and the syndecan-1 cytoplasmic domain controlled the shedding of the syndecan-1 extracellular domain. The work describes a mechanistic investigation into inside-to-outside syndecan signaling and highlights several gaps in our understanding of the relation between cell-surface receptors and proteases. In this Perspective, we summarize the current understanding of receptor interplay and identify the challenges that face investigators of adhesion- and growth factor–dependent signaling.

Emerging properties of adhesion complexes: what are they and what do they do?

The regulation of cell adhesion machinery is central to a wide variety of developmental and pathological processes and occurs primarily within integrin-associated adhesion complexes. Here, we review recent advances that have furthered our understanding of the composition, organisation, and dynamics of these complexes, and provide an updated view on their emerging functions. Key findings are that adhesion complexes contain both core and non-canonical components. As a result of the dramatic increase in the range of components observed in adhesion complexes by proteomics, we comment on newly emerging functions for adhesion signalling. We conclude that, from a cellular or tissue systems perspective, adhesion signalling should be viewed as an emergent property of both the core and non-canonical adhesion complex components.

The integrin cytoplasmic-tail motif EKQKVDLSTDC is sufficient to promote tumor cell invasion mediated by matrix metalloproteinase (MMP)-2 or MMP-9

Integrins promote cellular invasion through a combination of activities, including adhesion to an extracellular matrix ligand, which result in the generation of intracellular signals that lead to changes in cell behavior. Until now, there have been no data that identify a particular region of the cytoplasmic tail of integrin subunits as being responsible specifically for promoting the invasive activity of tumor cells. In this report, we show that amino acids with the sequence EKQKVDLSTDC, which are the C-terminal residues of the integrin β6 subunit, promote αvβ6-dependent invasion in a matrix metalloproteinase (MMP)-9-dependent fashion. This same peptide sequence, when expressed at the cytoplasmic end of the β3 integrin subunit, was able to enhance αvβ3-mediated invasive and enzymatic activity of tumor cells in an MMP-2-dependent fashion. Our results show that these 11 amino acids, when expressed at the C terminus of the β subunit, are responsible for regulating the activity of invasion-promoting degradative enzymes, whereas the specific MMP involved in this cellular behavior is dependent on the context of the remainder of the β integrin subunit.

Reduction of TSG101 protein has a negative impact on tumor cell growth.

TSG101 was defined originally as a tumor‐suppressor gene, raising the expectation that absence of the encoded protein should lead to increased tumor cell growth and, perhaps, increased tumor cell aggressiveness. We have used the RNA interference (RNAi) technique to downregulate TSG101 in PC3 (prostate cancer) and MDA‐MB‐231 (breast cancer) cells. An approximately 85% selective downregulation at the protein level was achieved in both cell lines over a period of 12 days as detected by Western blotting. This treatment resulted in inhibition of tumor cell growth, with a decreased level of TSG101 causing partial cell cycle arrest at the G1/S boundary and a reduction in the rate at which cells passed from G2 through mitosis and back into G1. In both cell lines, the percentage of cells in S‐phase was reduced significantly at day 4 after the TSG101 siRNA transfection (27% vs. 41% in MDA‐MB‐231 cells; 22% vs. 39% in PC3 cells). Additionally, RNAi‐mediated downregulation of TSG101 reduced the colony formation capacities of both cancer cell lines. Rather more surprisingly, TSG101 downregulation affected the migratory activity of the MDA‐MB‐231 cells, independent of any effect on proliferation. Thus, in a Transwell assay, after 4‐hr incubation, 36.0% of control MDA‐MB‐231 cells had migrated to the lower chamber vs. 7.3% of TSG101‐downregulated cells (p < 0.001; scrambled control, 36.5%). These results show that the TSG101 gene does not comply with the usual characteristics of a tumor‐suppressor gene; rather, its expression may be necessary for activities associated with aspects of tumor progression.