Trends in Cell Biology
ReviewGrowth control by intracellular tension and extracellular stiffness
Introduction
The extracellular microenvironment has profound effects on several cellular functions, including differentiation, apoptosis and proliferation. Although many studies have shown that localized release of soluble factors affects cellular function, the mixture of matrix proteins, proteoglycans and glucosaminoglycans that comprise the extracellular matrix (ECM) provide equally important cues that direct cellular decisions. Perhaps the newest idea about cellular control by the microenvironment is that the stiffness of the ECM (also referred to as its ‘compliance’) itself provides information to the cells. Several recent reviews have discussed the effects that ECM stiffness has on cell differentiation 1, 2. Here, we discuss data supporting the importance of the ECM, intracellular tension, cell shape, and ECM stiffness as regulators of integrin-dependent cell proliferation. Although we focus on studies showing that the ECM regulates progression through G1 phase, we note a recent study indicating that integrin-mediated adhesion can also regulate M phase [3].
Section snippets
Regulation of cyclin D1 expression by the ECM
cyclin D1 has been termed a ‘mitogen sensor’ because its expression is induced by many mitogenic factors, including growth factors, cytokines and hormones. However, our early studies showed that the induction of cyclin D1 expression is blocked when mitogen-stimulated cells are unable to attach to the substratum (for review, see [4]). Some ECM components, such as fibronectin, synergize with growth factors to induce the expression of cyclin D1 mRNA, whereas others, such as hyaluronan [5] and
cyclin D1 gene expression requires ECM-mediated sustained ERK activity
Studies using pharmacological MAP-ERK kinase (MEK) inhibitors and dominant–negative or constitutively active mutants of the Extracellular signal-regulated protein kinase (ERK) cascade indicate that ERK activity – probably ERK5 in addition to the prototypical ERK1 and ERK2 [8] – stimulates cyclin D1 expression in mid-G1 phase in many cell types (Figure 1; for review, see [9]). ERK activity stimulates the cyclin D1 promoter, whereas inhibition of ERK represses the cyclin D1 promoter;
FAK and cyclin D1 gene expression
Focal adhesion kinase (FAK) is the canonical mediator of integrin signals [11], and several reports have linked FAK to the induction of cyclin D1 (Figure 1). FAK is recruited to focal adhesions through its Focal adhesion targeting (FAT) domain and is autophosphorylated at Y397 upon ECM-induced integrin clustering. Phosphorylation of Y397 creates a binding site for Src and results in the Src-dependent tyrosine phosphorylation of FAK at several other sites, including Y576 and Y577–
ECM binding to integrins downregulates cip/kip cdk inhibitors
In addition to regulating expression of cyclin D1, the ECM regulates the expression of members of the cip/kip cdk inhibitor family (e.g. p21cip1 and p27kip1) which regulate activity of cdk2 in G1 phase and entry into S phase (for review, see in 4, 16). Incubating cells in suspension blocks integrin signaling and leads to an increase in the levels of p21cip1 and p27kip1. Skp2 is the substrate-targeting component of the E3 ubiquitin ligase complex that targets p27kip1 for degradation, and
Effects of intracellular tension, the actin cytoskeleton, and Rho family GTPases on adhesion-dependent signaling to the cell cycle
The actin cytoskeleton plays a major role in determining the degree of intracellular tension of a cell, and a high-tensional state is characterized by the appearance of actin stress fibers. Studies using actin-depolymerizing drugs have shown that sustained ERK activity and ERK-dependent cyclin D1 induction correlate with actin stress fiber formation and the consequent increase in intracellular tension (for review, see [4]). The Rho–Rho kinase pathway increases actin polymerization and cellular
Effects of cell spreading on integrin signaling and cell proliferation
Although the majority of studies of intracellular tension use either pharmacological inhibitors of actin polymerization or dominant–negative Rho GTPases to disrupt intracellular tension, an alternative approach is to control cell spreading. Cell spreading can be controlled by plating cells on different densities of matrix protein or by plating cells onto micropatterned, matrix protein-coated ‘islands’ of defined size; to date, most of the substrates have been rigid. Micropatterning enables
Modeling the effects of tissue and ECM compliance on integrin signaling and proliferation
Although actin-depolymerizing drugs and deliberate Rho–Rho kinase inhibition (e.g. with C3 toxin, dominant–negative constructs, RNA interference or Y27632) have been widely used to study the effects of integrin signaling on cellular function, these approaches probably result in much more severe changes in f-actin and Rho–Rho kinase activity than occur in vivo. Additionally, a pervasive shortcoming of these approaches is that the cells are cultured on non-deformable 2D substrata (i.e. culture
A working model for cell cycle control by intracellular tension and extracellular stiffness
Collectively, the results discussed above indicate that integrin-regulated cell cycle progression is not a binary process of ‘off’ or ‘on’ but rather a collection of signaling events with different compliance or tensional thresholds (Figure 2). For example, at the highest level of ECM compliance, FAK autophosphorylation is reduced, ERK activity is not sustained, cyclin D1 is not expressed, and cdk inhibitor expression is upregulated. This molecular signature, which results in G1 phase arrest,
Conclusions and future directions
The extracellular matrix is remodeled physiologically and also pathologically in diseases as diverse as fibrosis, cancer and atherosclerosis. The results to date indicate that changes in ECM stiffness affect cell morphology, integrin signaling, and the actin cytoskeleton, thereby facilitating control of the cell cycle. Given that different integrin-dependent signaling events have distinct compliance thresholds, the molecular composition of integrin signaling complexes at high and low tissue
Acknowledgements
Work in our laboratory is supported by grants from the National Institutes of Health.
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