Related Experiment Videos
SRC-dependent outside-in signalling is a key step in the process of autoregulation of beta2 integrins in
Paola Piccardoni1, Stefano Manarini, Lorenzo Federico
1Laboratory of Vascular Biology and Pharmacology, Consorzio Mario Negri Sud, Via Nazionale 1, 66030, Santa Maria Imbaro, Italy.
Abstract:
In human PMN (polymorphonuclear cells), challenged by P-selectin, the beta2-integrin Mac-1 (macrophage antigen-1) promoted the activation of the SRC (cellular homologue of Rous sarcoma virus oncogenic protein) family members HCK (haematopoietic cell kinase) and LYN (an SRC family protein tyrosine kinase) and phosphorylation of a P-110 (110 kDa protein). SRC kinase activity in turn was necessary for macrophage antigen-1-mediated adhesion [Piccardoni, Sideri, Manarini, Piccoli, Martelli, de Gaetano, Cerletti and Evangelista (2001) Blood 98, 108-116]. This suggested that an SRC-dependent outside-in signalling strengthens the beta2-integrin interaction with the ligand. To support this hypothesis further, in the present study, we used the monoclonal antibody KIM127 or manganese to lock beta2 integrins in a high-affinity state, and homotypic PMN adhesion was analysed to monitor beta2-integrin adhesive function. KIM127 or manganese induced PMN homotypic adhesion and P-110 phosphorylation. Both these processes were abolished by blocking antibodies against the common beta2 chain, by a combination of antibodies against alphaL and alphaM or by inhibitors of SRC activity. Confocal microscopy showed that activation epitopes were expressed by beta2 integrins co-localized with patches of F-actin at the adhesion sites. Blockade of SRC kinases or of actin polymerization prevented clustering of activated integrins as well as F-actin accumulation. FACS analysis showed that SRC inhibitors modified neither basal nor manganese-induced KIM127 binding. An SRC-dependent outside-in signalling initiated by beta2 integrins was also required for adhesion triggered by interleukin-8. These results confirm the hypothesis that an SRC-dependent outside-in signalling triggered by high affinity and ligand binding is necessary to stabilize beta2-integrin-mediated adhesion. Allowing clustering of activated integrins, SRC might link the high-affinity with the high-avidity state. Proline-rich tyrosine kinase-2 appears to be involved in this process.
Insights
SRC kinases are essential for stabilizing beta2-integrin-mediated adhesion in polymorphonuclear cells (PMN). This outside-in signaling pathway strengthens cell interactions by promoting integrin clustering and F-actin accumulation, crucial for immune responses.
Area of Science:
- Cellular Biology
- Immunology
- Molecular Signaling
Background:
- Beta2-integrin Mac-1 (macrophage antigen-1) activation is promoted by SRC family kinases (SFKs) like HCK and LYN in human polymorphonuclear cells (PMN).
- SRC kinase activity is critical for Mac-1-mediated adhesion, suggesting an SRC-dependent outside-in signaling pathway strengthens beta2-integrin-ligand interactions.
Purpose of the Study:
- To investigate the role of SRC-dependent outside-in signaling in stabilizing beta2-integrin-mediated adhesion.
- To determine if SRC kinases link high-affinity integrin states to high-avidity cell adhesion.
Main Methods:
- Utilized monoclonal antibody KIM127 or manganese to induce a high-affinity state in beta2 integrins.
- Analyzed homotypic PMN adhesion, P-110 phosphorylation, and F-actin accumulation.
- Employed blocking antibodies, SRC inhibitors, confocal microscopy, and FACS analysis.
Main Results:
- KIM127 or manganese induced PMN homotypic adhesion and P-110 phosphorylation, processes abolished by SRC inhibitors or blocking antibodies.
- SRC kinase and actin polymerization blockade prevented activated integrin clustering and F-actin accumulation.
- SRC-dependent outside-in signaling was also required for interleukin-8-triggered adhesion.
Conclusions:
- SRC-dependent outside-in signaling, triggered by high-affinity and ligand binding, is necessary for stabilizing beta2-integrin-mediated adhesion.
- SRC kinases facilitate the transition from high-affinity to high-avidity states by promoting activated integrin clustering.
- Proline-rich tyrosine kinase-2 is implicated in this signaling pathway.
Related Concept Videos
Autocrine Signaling
Autocrine Signaling in Macrophages
Under normal physiological conditions, autocrine signaling is essential for maintaining homeostasis. This process is well characterized in...
Intracellular Signaling Affects Focal Adhesions
Some...
Activation of Integrins
In "outside-in signaling," external factors in the extracellular space bind to exposed ligand binding sites on integrins. This causes the inactive protein to undergo a conformational change to become active. Integrins are often clustered on the cell membrane. Repetitive and regularly spaced ligand binding...
Autoregulation of Blood Flow
Chemical Signaling in Autoregulation
Chemical signaling operates at the precapillary sphincter level, inciting either contraction or relaxation....
Paracrine Signaling
Overview of Cell Signaling
Cells respond to many types of information, often through receptor proteins positioned on the membrane. For example, skin cells respond to and transmit touch...