Shaping Pancreatic β-Cell Differentiation and Functioning: The Influence of Mechanotransduction

Abstract

Embryonic and pluripotent stem cells hold great promise in generating β-cells for both replacing medicine and novel therapeutic discoveries in diabetes mellitus. However, their differentiation in vitro is still inefficient, and functional studies reveal that most of these β-like cells still fail to fully mirror the adult β-cell physiology. For their proper growth and functioning, β-cells require a very specific environment, the islet niche, which provides a myriad of chemical and physical signals. While the nature and effects of chemical stimuli have been widely characterized, less is known about the mechanical signals. We here review the current status of knowledge of biophysical cues provided by the niche where β-cells normally live and differentiate, and we underline the possible machinery designated for mechanotransduction in β-cells. Although the regulatory mechanisms remain poorly understood, the analysis reveals that β-cells are equipped with all mechanosensors and signaling proteins actively involved in mechanotransduction in other cell types, and they respond to mechanical cues by changing their behavior. By engineering microenvironments mirroring the biophysical niche properties it is possible to elucidate the β-cell mechanotransductive-regulatory mechanisms and to harness them for the promotion of β-cell differentiation capacity in vitro.

Keywords: YAP/TAZ; actin; diabetes; integrin; islet of Langerhans; mechanotransduction; nanotopography; stem cells; β-cells.

Figure 1

The islet niche and the extracellular factors influencing β-cell development, differentiation, and function. The islet niche is a complex and multi-factorial microenvironment that is characterized by the presence of different cells, a specific extracellular matrix, and several chemical, metabolic, and physical cues. The interactions between β-cells and their environment are extremely dynamic and bidirectional, as β-cells perceive the extracellular signals and respond to them, thus shaping the niche architecture.

Figure 2

Mechanobiology in the islet (A) Mechanosensing and mechanotransduction in β-cells. Mechanical cues (substrate stiffness, topography and geometry, and fluid shear stress) are perceived by several mechanosensors (integrins, cadherins, mechanosensitive ion channels, primary cilia, and glycocalyx) located at the plasma membrane of β-cells. We are focusing here on integrin-based mechanotransduction. ECM physical properties are sensed by integrins and interpreted by integrin-mediated mechanotransduction, involving actin cytoskeletal actions, and the formation of integrin adhesion complexes (IAC). The latter can reach different maturation and signaling stages (from nascent adhesions, via focal complexes to focal adhesions). The dynamic composition and dimensions of IACs depend directly on the biophysical characteristics of the microenvironment that the cell encounters and can simultaneously regulate two signaling transduction pathways, differing in the timing of cellular responses. The ‘fast’ mechanoresponse (left side, time scale: milliseconds) is directly mediated by the spatial reorganization of the acto-myosin cytoskeleton, which generates modulations in tension and causes the modification of nuclear architecture and mitochondria dynamics. The slower mechanoresponse (right side, time scale: seconds to hours) is mediated by complex cascades of protein interactions and phosphorylations, which culminate with mechanosensitive transcription factors (TF, e.g., YAP/TAZ) stabilization and shuttling to the nucleus where they control gene transcription and shape the cellular program. (B) Mechanotransduction at the plasma membrane. The interaction of integrins with the ECM triggers the initial connection to the actin filaments (F-actin) of the cytoskeleton (via talin) and the engagement of the molecular clutch (ECM/integrin/talin/F-actin linkage) to the acto-myosin-generated forces in the nascent adhesions. The biophysical features of the ECM (in terms of the rigidity and nanometric spatial organization of the adhesion sites) determine whether these initial structures will either disintegrate, or (as depicted in the scheme) reinforce and recruit adaptor proteins (e.g., vinculin and paxillin), actin regulators (e.g., α-actinin), and signaling molecules (FAK—Focal adhesion kinase) to the nascent adhesions. This reinforcement and protein recruitment leads to a maturation of the structure (e.g., to focal adhesions) and its transformation into a signaling hub, which influences actin cytoskeletal dynamics, such as actin polymerization and the generation of acto-myosin contraction, by RhoA/ROCK pathway activation and other mechanosensitive signaling pathways (e.g., channels, YAP/TAZ). (C) Mechanotransduction at the nucleus. The state of cytoskeletal organization and tension, regulated by the acto-myosin fiber contraction, impacts the nuclear envelope (which is connected to the cytoskeleton via the LINC complex) and deforms its architecture and permeability to control chromatin condensation and gene activation.

Figure 3

β-cells sense the ECM-mimicking nanotopography and activate a mechanotransduction-dependent program, which promotes their differentiation. Human islets of Langerhans grown on flat (A–C) or nanostructured (D–F) zirconia for 20 days were stained with DAPI (4′,6-diamidino-2-phenylindole) (blue) and for actin (red), and vinculin (green) to visualize the modulation of the mechanotransduction pathway in (B,E); or with DAPI (blue) and insulin (green) to ascertain the β-cell phenotype (C,F). (A–C) Cell–matrix interactions predominate in islet cells grown on flat substrates. β-cells present few insulin granules and are scattered on this substrate. (D–F) Cells grown on the nanotopographical substrates instead adopt a round shape, which favors the establishment of cell–cell contacts and the organization in islet-like clusters where β-cells are full of insulin granules. Note also the different nuclear shapes and sizes of the cells grown on flat or nanostructured substrates [64].

Figure 4

YAP signaling controls pancreatic β-cell maturation. The fate of bipotent pancreatic progenitor cells is under the control of the YAP transcription factor. Its activation promotes progenitor cell differentiation toward a ductal fate and blunt endocrinogenesis, through suppression of NGN3. Its inactivation (via modification of ECM stiffness, nanotopography, geometry, exposure to laminin, or pharmacological inhibitors) is required to allow NGN3 expression and differentiation toward an endocrine cell fate, characterized by expression of PDX1, Neurogenic differentiation 1 (NEUROD1) and MAFA transcription factors, as well as insulin.