Integrins, the best characterized cell-ECM mechanoreceptors, are composed of non-covalently linked heterodimeric α and β subunits. Twenty-four distinct integrin subtypes have been identified in mammals and are made up of eighteen α subunits (α1–11, αV, αIIb, αL, αM, αX, αD and αE) and eight β subunits (β1–8) [
11,
22]. Each integrin subunit possesses a large extracellular domain, a single transmembrane domain, and a short cytoplasmic tail (except for β4). The α subunits allow integrins to selectively bind to distinct motifs of ECM proteins (Arg-Gly-Asp (RGD) motif, collagen and laminin) [
11], whereas the β subunits are responsible for the interaction of integrin with the actomyosin cytoskeleton through numerous anchoring proteins, connecting multiple signaling pathways [
23]. Heterodimerization of integrin often takes place within the cell before presentation on the cell surface through the interaction between the β-propeller domain of the α subunit and the hybrid domain of the β subunit [
24].
2.1. Integrin Activation
Inactive integrin heterodimers are usually found in a bent conformation and possess low affinity for their ligands. Once activated, integrins undergo rapid conformational changes to become extended with increased ligand affinity. Intracellularly, the conformational changes of integrins can be accomplished by binding of intracellular activators (e.g., talin and kindlin) [
25,
26]. Both activators possess a FERM (4.1/ezrin/radixin/moesin) domain that enables them to bind to the β subunit cytoplasmic tails of integrins. However, whether the activators work together or independently is not yet fully understood. Integrins span the cell membrane and are exposed on both the inside and outside of the cell. Therefore, the activation of integrins can take place in both sides of the membrane, consequently integrins transduce signals bidirectionally across the membrane [
27,
28]. Integrin activation can occur by biochemical or mechanical ligands. Through their extracellular domains, integrins can be activated by mechanical stimuli from within the ECM and stromal cells, or by binding to ECM proteins, such as collagen, fibronectin, fibrinogen, laminin and vitronectin as their ligands. Integrin clustering is regulated not only by ECM ligands, but by other extracellular integrin interacting partners (e.g., galectins and tetraspanins) as they bind to the extracellular domain of integrins in a ligand-independent manner [
21]. Moreover, biochemical and mechanical ligands can be applied reciprocally to activate integrins. In support of this hypothesis, it was found that ligand-induced mechanical force by steered molecular dynamics (SMD) stimulation accelerates hinge-angle opening, resulting in the transition of integrins from an inactive to an active conformation with high ligand-binding affinity [
29].
Activated integrins exhibit high affinity to extracellular ligands, leading to integrin clustering at the cell membrane which stabilizes the cell-ECM interaction. Consequently, extracellular signals are transduced through integrins into the cell, known as outside-in signaling that affects various cellular behaviors, including motility, spreading, migration, growth, survival, proliferation and differentiation [
30,
31]. This signal transduction cannot be solely governed by integrins as they are not capable of phosphorylating or dephosphorylating other molecules in the signaling cascade. Instead, recruited kinases (e.g., FAK, ILK and Src) upon integrin activation carry out the task to deliver the signals to the downstream molecules.
2.2. Ligand Specificity and Diversity
Distinct extracellular domains of integrin α subunits give each integrin the binding specificity to proteins of the ECM [
24]. For instance, collagen binding integrins consist of α1, α2, α10 or α11 subunits, which heterodimerize only with the β1 subunit, and recognize the same motif, GFOGER. Alternatively, α3, α6, α7 subunits belong to a subgroup of integrins that bind to laminin [
11,
32]. The number of interacting ECM ligands also varies amongst different integrins, for example, integrin α5β1 recognizes primarily fibronectin, whereas αvβ3 binds to multiple RGD containing proteins such as fibronectin and vitronectin [
33]. Even when they interact with the same ligands, the integrins can recognize them in a different manner. For example, although integrins α5β1 and α4β1 are known to bind to fibronectin, their recognition sequences in fibronectin are different. Integrin α5β1 recognizes the RGD sequence in fibronectin, whereas integrin α4β1 recognizes EILDV and REDV sequences.
A different combination of integrin subunits can have a distinct impact on the same cellular response. For instance, in the context of glomerulosclerosis, caused by abnormal collagen deposition in the glomerulus, integrins α1β1 and α2β1 play different roles. Whereas integrin α2β1 binds to collagen type I and positively regulates collagen synthesis, integrin α1β1 interacts with collagen type IV to negatively modulate the synthesis of collagen. As a result, integrin α2β1 exacerbates the condition, but integrin α1β1 protects from glomerular injury, indicating that expression of a subset of integrins can result in contradictory cellular responses based on its ECM ligand [
34,
35]. Another example of different responses to similar subunit combinations involves αv subunit-containing integrins that bind to the RGD motif. Five different β subunits (β1, β3, β5, β6 and β8) are known to form heterodimers with the αv subunit, but different combinations can give rise to diverse cellular responses [
36]. For example, integrin αvβ3, the most studied αv subunit-containing integrin, binds to RGD motif containing proteins (e.g., vitronectin, fibronectin, fibrinogen, and von Willebrand factor), collagen, laminin and also interacts with vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor (PDGF), fibroblast growth factor-2 (FGF2), matrix metalloproteinase-2 (MMP-2) and insulin [
37,
38]. Integrin αvβ3 is broadly expressed in different types of cells such as endothelial cells, osteoclasts and blood cells. Notably, the expression of the receptor is high in tumors including activated tumor endothelial cells, glioblastomas, malignant melanomas, breast, lung, pancreatic and prostate carcinomas, but not in resting endothelial cells and normal organ systems [
39,
40,
41,
42,
43]. Furthermore, integrin αvβ3, in association with vitronectin, has been found to facilitate the differentiation of prostate and breast cancer stem cells (CSCs) and subsequently promote tumor formation [
44]. Diverse types of integrins have been implicated in cancer progression (see
Table 1).
Integrin αvβ5, another αv subunit-containing integrin, is expressed in glioblastomas together with integrin αvβ3 [
45]. In addition, the interaction of integrin αvβ5 with TGFBI (transforming growth factor beta-induced), an ECM interacting protein that contains RGD motif, promotes the activation of FAK signaling pathway, consequently enhancing glycolysis and invasiveness in pancreatic cancer cells [
52].
2.3. Integrins in Mechanotransduction
Many types of mechanical cues are present in the ECM, including substrate rigidity, fluid shear stress, hydrostatic pressure, tensile and compressive forces, and diverse integrin subtypes are involved in mechanosensing and force transmission.
Tissues exhibit different mechanical properties depending on their locations and functions. For example, brain tissue has an elastic modulus of several hundred pascal whereas that of skeletal muscle is approximately 100 kPa [
73], and most solid tumors possess considerably higher stiffness than the surrounding normal tissues [
74,
75]. The gradual stiffening of the tumor stroma arises from deposition and remodeling of the ECM and is believed to promote cancer progression and metastasis [
76,
77,
78]. Changes in matrix stiffness can be recognized by cells, and more rigid substrates often result in greater cellular forces exerted onto the matrix (traction force) along with increased stress fibers and larger focal adhesions [
79]. As a result, the mechanical balance is created by the contractility of the cell and the mechanical properties of the ECM. Various biophysical techniques have been developed to study the diverse mechanical properties of cells in mechanotransduction, including traction force microscopy (TFM), magnetic twisting/pulling cytometry (MTC/MPC), optical tweezers, atomic force microscopy (AFM) and shear flow microfluidic devices [
80].
Using TFM and surface plasmon resonance to measure forces exerted by cells and binding dynamics of integrins, it has been determined that binding of breast myoepithelial cells to fibronectin through integrins α5β1 or αvβ6 allows them to sense and adapt to different tissue stiffness. While integrin α5β1 is in charge of responding to the stiffness in healthy tissue, αvβ6 plays the corresponding role in malignant tissue [
81]. Our lab has used TFM and mechanosensing experiments to understand the mechanisms by which cells regulate the production of traction force and mechanosensing in response to different types of mechanical stimuli. TFM analysis enabled us to identify calpain 4 as a key regulator in the generation of traction force during migration of embryoninc fibroblasts [
82]. Calpain 4 is the small regulatory subunit that forms a heterodimer with one of two large catalytic subunits (calpain 1 and calpain 2) in the calpain family of proteases, and calpains have been implicated in integrin-mediated cell adhesion and migration [
83,
84,
85]. The function of calpain 4 in regulating cellular traction forces was found to be independent of proteolytic activities of catalytic subunits [
82]. On the other hand, the two catalytic subunits and the regulatory subunit are necessary for cells to respond to changes in substrate topography and externally applied mechanical stimulation, suggesting distinct regulation of mechanotransduction in the cell in response to different types of mechanical cues. We have also observed an inverse relationship between traction stress and metastatic capacity of breast cancer cells. Traction stress of four murine mammary carcinoma cell lines with varying metastatic capacities were tested, and force production decreased as the metastatic potential and capacity increased [
86]. In the same manner, adhesion strength, the number of focal adhesion proteins and the amount of active integrin β1 were also inversely correlated with metastatic ability of cells. It is not surprising that focal adhesion proteins and integrins are closely involved in the force generation and transduction, given that traction forces are generated by the cell and transmitted to the ECM.
The ability of cells to sense stiffness can also be attributed to the integrins. For example, the αvβ3 integrin has been found to recognize the stiffness of fibronectin-coated substrates. Fibroblasts incubated with anti-αvβ3 monoclonal antibody that blocks the interaction between integrin αvβ3 and fibronectin failed to sense the rigidity of fibronectin matrix [
87]. Similarly, cells lacking RPTPα, a receptor-like protein tyrosine phosphatase, lost the capability to sense the matrix stiffness. This indicates the function of integrin αvβ3 in association with RPTPα in sensing the rigidity of fibronectin matrix at the leading edge during early spreading.
Force-dependent conformational changes in integrin αLβ2 have also been observed and correlate with dissociation from intercellular adhesion molecule 1 (ICAM-1). Increasing tensile forces, using a biomembrane force probe (BFP), applied on the catch bond between integrin αLβ2 and ICAM-1 lead to a more active conformation with higher affinity for its ligand [
88]. Likewise, bending and unbending conformational changes of integrin αvβ3 was found to be dependent on tensile forces [
89,
90]. Force-dependent binding properties of integrin αvβ3 have been identified by using optical tweezers and force-ramp assays, demonstrating that pulling forces regulate the interaction between integrin αvβ3 and Thy-1 and a slip bond behavior of Thy-1/αvβ3 interaction [
91]. As one such approach to understand the influence of tugging forces generated within the tumor stroma on cancer cells, an in vitro mechano-invasion assay was designed to mimic the TME where cancer cells are subjected to mechanical stimuli produced by neighboring highly contractile cells such as CAFs possessing myofibroblastic properties [
92]. In this assay, random transient tugging forces are applied to the cells seeded on the collagen type I/fibronectin substrate. The forces on the cancer cells are created by carboxylated paramagnetic microbeads, covalently linked to ECM proteins and randomly positioned, as a rotating magnet twitches the beads surrounding the cells. The applied transient mechanical stimulation led to enhanced invasiveness of highly invasive human fibrosarcoma cells in a fibronectin-dependent manner, but no significant effect was observed in poorly invasive or normal cells. In addition, the pro-invasive effect in response to tugging forces was found to be dependent on cofilin, a known regulator of invadopodia maturation [
71]. Forty-six genes were identified as differentially expressed in mechanically stimulated cells, including several downregulated genes. Surprisingly,
ITGB3 (encoding the known mechanoreceptor integrin β3) was found to be downregulated. Integrin β3 binds to fibronectin, a required component needed to increase cell invasion in response to mechanical stimulation [
92]. Downregulated integrin β3 expression upon transient mechanical stimulation augments the amount of active cofilin and enhances enzymatic activity of MMP-2 localized in invadopodia, thus promoting the maturation of invadopodia and the invasion of metastatic cells [
71].
Integrins also function as mechanoreceptors in response to shear stress. For instance, integrin αvβ3 and integrins containing β1 and β5 subunit can recognize increased shear stress from in vitro flow channels, leading to their associations with the adaptor protein Shc in bovine aortic endothelial cells (BAECs) [
93]. Integrin αvβ3 activation by shear stress also leads to enhanced affinity to ECM proteins in BAECs [
94]. In further studies, breast cancer cell attachment through interaction with platelets was attributed to the activation status of integrin αvβ3 under blood flow conditions with shear stress [
46]. Integrin αvβ3 activation further elicits the metastatic potential in breast cancer cells [
46]. Using single-bond BFP and a multiple bond flow chamber, it has been demonstrated that tensile force and shear stress regulate the interaction between integrin α4β1 and its main ligand vascular cell adhesion molecule-1 (VCAM-1) [
95].