Alpha5 Antibodies Research Articles

Tumor cell invasiveness correlates with changes in integrin expression and localization

In nontumorigenic mammary epithelial cells (EpH4), transforming growth factor-beta (TGFbeta1) causes cell cycle arrest/apoptosis, but induces epitheliomesenchymal transition (EMT) in Ha-Ras-transformed EpH4 cells (EpRas). EMT is closely correlated with late-stage tumor progression and results in fibroblastic, migratory cells displaying a mesenchymal gene expression program (FibRas). EpRas and FibRas cells showed strongly increased cell substrate adhesion to fibronectin, collagens I/IV and laminin 1. Furthermore, Ras transformation caused enhanced or de-novo expression of the integrin subunits beta1, alpha2 and alpha3, or alpha5 and alpha6, respectively, the latter subunits being even more strongly expressed in FibRas cells. Importantly, polarized EpRas cells expressed integrin subunits beta1 and alpha6 at distinct (apical and lateral) membrane domains, while FibRas cells coexpressed these integrins and alpha5 at the entire plasma membrane. During EMT, EpRas cells formed an alpha5beta1 complex and deposited its ligand fibronectin into the extracellular matrix. Function-blocking alpha5 antibodies attenuated migration, and caused massive apoptosis in EpRas cells undergoing TGFbeta1-induced EMT in collagen gels, but failed to affect EpRas- or FibRas-derived structures. We conclude that functional alpha5beta1 integrin is centrally implicated in EMT induction. Importantly, FibRas cells also failed to deposit the alpha6beta4 ligand laminin 5, suggesting that alpha6beta4 is no longer functional after EMT and replaced by mesenchymal integrins such as alpha5beta1.

Evidence that integrins contribute to multiple stages in the consolidation of long term potentiation in rat hippocampus

Three structurally distinct groups of antagonists were used to test the hypothesis that integrin adhesion receptors play an essential role in consolidating (stabilizing) long term potentiation of the Schaffer collaterals in rat hippocampus. Comparisons were made of percent potentiation at antagonist-treated versus control sites within CA1 stratum radiatum of the same hippocampal slice. Function blocking antibodies against the alpha5 subunit of the fibronectin receptor had no effect on baseline responses or initial potentiation but resulted in a >30% reduction, relative to within-slice control long term potentiation, 45 min later. Larger reductions were recorded in separate experiments continued for 4 h after the induction of potentiation. Alpha(v) and alpha2 subunit antibodies did not reliably affect the stabilization of potentiation. An antagonist peptide with preference for beta1 integrins produced a slowly developing decline of the type seen with alpha5 antibodies. A cyclic peptide antagonist reduced potentiation within 10 min of induction and caused an almost 40% decrease over 45 min. Two disintegrins (snake toxins that potently block integrins) were very effective in preventing the consolidation of long term potentiation: echistatin reduced potentiation by >70%, while triflavin caused ∼50% decrease. The suppressing effects of echistatin were concentration-dependent, obtained with treatment after induction, and much more rapid than the effects of antibodies. Rapid declines in potentiation were particularly evident when the two disintegrins were applied together. These results indicate that hippocampal fibronectin receptors (alpha5/beta1 integrin) contribute importantly to a slowly developing phase of long term potentiation consolidation. They also suggest that other integrins are critical to aspects of consolidation occurring in the first few minutes after induction.

Evidence for integrin receptor involvement in megakaryocyte-fibroblast interaction: a possible pathomechanism for the evolution of myelofibrosis.

Megakaryocytes are assumed to be functionally linked with the evolution of myelofibrosis, complicating chronic myeloproliferative disorders. It has already been shown that megakaryocytes will promote fibroblast growth in vitro when in spatial proximity. Here, we demonstrate that the integrin receptors alpha3beta1 and alpha5beta1 are involved in this megakaryocyte-fibroblast interaction. Upon addition of anti-alpha3 and -alpha5 antibodies to megakaryocyte-fibroblast cocultures, fibroblast growth was significantly impaired, and megakaryocyte attachment to the fibroblast feederlayer was significantly reduced. Unilateral blocking of megakaryocytes with anti-alpha3 or -alpha5 antibodies resulted in a suppression of adhesion, probably reflecting the prominent function of fibronectin receptors on the megakaryocyte surface. Moreover, the oligopeptide RGDS (Asp-Gly-Asp-Ser) caused a significant reduction of fibroblast growth as well as megakaryocyte adhesion. This feature reinforces that fibronectin receptors are involved. In addition, fibroblast proliferation was impaired by the application of fibronectin antibodies recognizing the cell-binding domain. However, no effect was observable with respect to megakaryocyte adhesion. In conclusion, our in vitro studies demonstrate the involvement of beta1-integrins, in particular the fibronectin receptor in the megakaryocyte-dependent fibroblast proliferation and therefore suggest a pivotal role of megakaryocytes in the complex pathomechanism causing myelofibrosis.

Extracellular signal-regulated kinase and c-Jun NH2-terminal kinase activation by mechanical stretch is integrin-dependent and matrix-specific in rat cardiac fibroblasts.

Integrins, which connect the cytoskeleton to the extracellular matrix and mediate a variety of signaling cascades, may transduce mechanical stimuli into biochemical signals. We studied integrin- and matrix-dependent activation of extracellular signal-regulated kinase (ERK2), c-Jun NH2-terminal kinase (JNK1), and p38 in response to 4% static biaxial stretch in rat cardiac fibroblasts. ERK2 and JNK1, but not p38, were rapidly activated by stretch when the fibroblasts were allowed to synthesize their own matrices. When the cells were limited to specific matrix substrates, ERK2 and JNK1 were differentially activated: ERK2 was only activated when the cells were plated on fibronectin, while JNK1 was activated when the cells were plated on fibronectin, vitronectin, or laminin. Plating cells on collagen before stretching did not activate either kinase. Adhesion to all matrices was integrin-dependent because it could be blocked by inhibitors of specific integrins. ERK2 activation could be blocked with a combination of anti-alpha4 and -alpha5 antibodies and an arginine-glycine-aspartic acid (RGD) peptide, while the antibodies or peptide used separately failed to block ERK2 activation. This result suggests that at least two integrins, alpha4beta1 and an RGD-directed, non-alpha5beta1 integrin, activate ERK2 in response to mechanical stimulation. Activation of JNK1 could not be blocked with the inhibitors, suggesting that an RGD-independent integrin or integrins other than alpha4beta1 can activate JNK1 in cells adherent to fibronectin. This study demonstrates that integrins act as mechanotransducers, providing insight into potential mechanisms for in vivo responses to mechanical stimuli.