The fibronectin-binding integrins α5β1 and αvβ3 differentially modulate RhoA–GTP loading, organization of cell matrix adhesions, and fibronectin fibrillogenesis

Danen, Erik H.J.; Sonneveld, Petra; Brakebusch, Cord; Fässler, Reinhard; Sonnenberg, Arnoud

doi:10.1083/jcb.200205014

Cited by 302 publications

(329 citation statements)

References 78 publications

(115 reference statements)

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“…One method by which fibroblasts respond to external signals is through integrins (41) and the most common integrins found in focal adhesions contain the b1 and b3 subunits (42,43). To determine whether these integrin subunits mediate fibroblast activation in response to exogenous LOXL2, we used b1 and b3 function-blocking antibodies.…”

Section: Loxl2 Mediates Fibroblast Activation Through B3 Integrinsmentioning

confidence: 99%

Tumor-Secreted LOXL2 Activates Fibroblasts through FAK Signaling

Barker

Bird

Lang

et al. 2013

Molecular Cancer Research

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Cancer-associated fibroblasts enhance cancer progression when activated by tumor cells through mechanisms not yet fully understood. Blocking mammary tumor cell-derived lysyl oxidase-like 2 (LOXL2) significantly inhibited mammary tumor cell invasion and metastasis in transgenic and orthotopic mouse models. Here, we discovered that tumor-derived LOXL2 directly activated stromal fibroblasts in the tumor microenvironment. Genetic manipulation or antibody inhibition of LOXL2 in orthotopically grown mammary tumors reduced the expression of a-smooth muscle actin (a-SMA). Using a marker for reticular fibroblasts, it was determined that expression of a-SMA was localized to fibroblasts recruited from the host tissue. This marker also revealed that the matrix present in tumors with reduced levels of LOXL2 was more scattered compared with control tumors which exhibited matrices with dense, parallel alignments. Importantly, in vitro assays revealed that tumor-derived LOXL2 and a recombinant LOXL2 protein induced fibroblast branching on collagen matrices, as well as increased fibroblast-mediated collagen contraction and invasion of fibroblasts through extracellular matrix. Moreover, LOXL2 induced the expression of a-SMA in fibroblasts grown on collagen matrices. Mechanistically, it was determined that LOXL2 activated fibroblasts through integrinmediated focal adhesion kinase activation. These results indicate that inhibition of LOXL2 in tumors not only reduces tumor cell invasion but also attenuates the activation of host cells in the tumor microenvironment.Implications: These findings reveal new insight into the mechanisms of fibroblast activation, a novel function of LOXL2, and further highlight the importance of generating LOXL2-targeted therapies for the prevention of tumor progression and metastasis.

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Section: Loxl2 Mediates Fibroblast Activation Through B3 Integrinsmentioning

confidence: 99%

Tumor-Secreted LOXL2 Activates Fibroblasts through FAK Signaling

Barker

Bird

Lang

et al. 2013

Molecular Cancer Research

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“…Moreover, an initial dip in Rho activity has been observed when fibroblasts are plated on fibronectin (23) or upon treating them in suspension with soluble RGD peptides (34) known to engage ␣ 5 ␤ 1 integrin. Reports also indicate that ␤ 3 and ␤ 1 integrins could lead to different responses, but the effect seems to depend on the cell type (37,38). However, in all cases, activation of the receptors was achieved using ECM-derived ligands.…”

Section: Discussionmentioning

confidence: 99%

Aggregation of Integrins and RhoA Activation Are Required for Thy-1-induced Morphological Changes in Astrocytes

Avalos

Arthur

Schneider

et al. 2004

Journal of Biological Chemistry

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Thy-1, a cell adhesion molecule abundantly expressed in mammalian neurons, binds to a ␤ 3 -containing integrin on astrocytes and thereby stimulates the assembly of focal adhesions and stress fibers. Such events lead to morphological changes in astrocytes that resemble those occurring upon injury in the brain. Extracellular matrix proteins, typical integrin ligands, bind to integrins and promote receptor clustering as well as signal transduction events that involve small G proteins and cytoskeletal changes. Here we investigated the possibility that the cell surface protein Thy-1, when interacting with a ␤ 3 -containing integrin on astrocytes, could trigger signaling events similar to those generated by extracellular matrix proteins. DI-TNC 1 astrocytes were stimulated with Thy-1-Fc immobilized on beads, and increased RhoA activity was confirmed using an affinity precipitation assay. The effect of various inhibitors on the cellular response was also studied. The presence of Y-27632, an inhibitor of Rho kinase (p160ROCK), a key downstream effector of RhoA, significantly reduced focal adhesion and stress fiber formation induced by Thy-1. Similar effects were obtained when astrocytes were treated with C3 transferase, an inhibitor of RhoA. Alternatively, astrocytes were transfected with an expression vector encoding fusion proteins of enhanced green fluorescent protein with either the Rho-binding domain of Rhotekin, which blocks RhoA function, or the dominantnegative N19RhoA mutant. In both cases, Thy-1-induced focal adhesion formation was inhibited. Furthermore, we observed that RhoA activity after stimulation with soluble Thy-1-Fc molecule was augmented upon further cross-linking using protein ASepharose beads. The same was shown by crosslinking ␤ 3 -containing integrin with anti-␤ 3 antibodies. Together, these results indicate that Thy-1-mediated astrocyte stimulation depended on ␤ 3 integrin clustering and the resulting increase in RhoA activity.Focal adhesions (FAs) 1 mediate strong adhesion to the substrate and anchor actin stress fibers (SFs) through a supramolecular complex that consists of many different cytoskeletal as well as signaling molecules (reviewed in Refs. 1 and 2). The unique features of FAs suggest that they are employed as mechanosensors to inform cells about the immediate environment. As such, FAs are implicated in transmembrane crosstalk between extracellular matrix (ECM) proteins and intracellular signal transduction pathways via integrin receptors (reviewed in Refs. 3 and 4).The interaction between ECM components and integrins leads to reorganization of the cytoskeleton and regulation of cell migration. Such events have been most extensively studied in fibroblasts (reviewed in Ref. 5) and linked there to activation of small GTPases belonging to the Rho family of monomeric 20 -30-kDa GTP-binding proteins. Like other members of the Ras superfamily, Rho acts as a molecular switch, cycling between an active GTP-bound state and an inactive, GDP-bound state (reviewed in Refs. 6 and 7). The best-cha...

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“…Enlarging on this last point, and as stated above, a growing set of proteins associated with focal adhesions, including the integrins, FAK, and others have been shown to be engaged in a reciprocal regulatory dialog with RhoA in control of cell attachment and cell motility during interphase (Flinn and Ridley, 1996;Arthur et al, 2000;Ren et al, 2000;Schwartz and Shattil, 2000;Sinnett-Smith et al, 2001;Arthur et al, 2002;Danen et al, 2002). FAK (Ϫ/Ϫ) null cells are nonmotile and excessively rounded; however, inhibition of p160ROCK in FAK(Ϫ/Ϫ) cells reverses rounding, implying FAK functions are dependent on RhoA (Chen et al, 2002).…”

Section: Discussionmentioning

confidence: 99%

Deregulation of HEF1 Impairs M-Phase Progression by Disrupting the RhoA Activation Cycle

Dadke

Jarnik

Pugacheva

et al. 2006

MBoC

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The focal adhesion-associated signaling protein HEF1 undergoes a striking relocalization to the spindle at mitosis, but a function for HEF1 in mitotic signaling has not been demonstrated. We here report that overexpression of HEF1 leads to failure of cells to progress through cytokinesis, whereas depletion of HEF1 by small interfering RNA (siRNA) leads to defects earlier in M phase before cleavage furrow formation. These defects can be explained mechanistically by our determination that HEF1 regulates the activation cycle of RhoA. Inactivation of RhoA has long been known to be required for cytokinesis, whereas it has recently been determined that activation of RhoA at the entry to M phase is required for cellular rounding. We find that increased HEF1 sustains RhoA activation, whereas depleted HEF1 by siRNA reduces RhoA activation. Furthermore, we demonstrate that chemical inhibition of RhoA is sufficient to reverse HEF1-dependent cellular arrest at cytokinesis. Finally, we demonstrate that HEF1 associates with the RhoA-GTP exchange factor ECT2, an orthologue of the Drosophila cytokinetic regulator Pebble, providing a direct means for HEF1 control of RhoA. We conclude that HEF1 is a novel component of the cell division control machinery and that HEF1 activity impacts division as well as cell attachment signaling events. INTRODUCTIONAs points of structural linkage between the extracellular matrix (ECM) and the intracellular cytoskeleton, focal adhesions possess a complex function. For example, during migration, cells must rapidly break down and reform adhesions with the ECM, providing force for propulsion (Lauffenburger and Horwitz, 1996). At mitotic entry, cultured cells round up and decrease adhesion to the ECM; at mitotic exit, basal attachments reassemble and contribute to the force generation required for efficient progress through cytokinesis and reentry into G 1 . In interphase cells, the formation of novel focal adhesion-ECM interactions can specify cellular differentiation by activating specific signaling cascades culminating in the induction of differentiationpromoting transcription factors, and in parallel enforce removal from the cell cycle (Boudreau and Bissell, 1998). In many cell types, sustained loss of adhesion is a sufficient stimulus to induce apoptosis (anoikis) (Frisch and Francis, 1994), a surveillance mechanism against cancer, inhibiting the formation of micrometastases. Hence, one frequent effect of oncogenic transformation is the circumvention of the adhesion-viability coupling, leading to acquisition by cancer cells of the ability to grow in an anchorage-independent manner (Schwartz, 1997). Based on these diverse biological roles, there has been considerable research effort directed at elucidating the signaling role of focal adhesion-associated proteins (Schlaepfer et al., 1999).HEF1, p130Cas, and Efs/Sin define the Cas family of proteins Bouton et al., 2001). In interphase cells, Cas proteins predominantly localize to focal adhesions. During initial integrin engagement, induced by cell a...

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The fibronectin-binding integrins α5β1 and αvβ3 differentially modulate RhoA–GTP loading, organization of cell matrix adhesions, and fibronectin fibrillogenesis

Cited by 302 publications

References 78 publications

Tumor-Secreted LOXL2 Activates Fibroblasts through FAK Signaling

Tumor-Secreted LOXL2 Activates Fibroblasts through FAK Signaling

Aggregation of Integrins and RhoA Activation Are Required for Thy-1-induced Morphological Changes in Astrocytes

Deregulation of HEF1 Impairs M-Phase Progression by Disrupting the RhoA Activation Cycle

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