Extracellular matrix structure and nano-mechanics determine megakaryocyte function

Abstract

Cell interactions with matrices via specific receptors control many functions, with chemistry, physics, and membrane elasticity as fundamental elements of the processes involved. Little is known about how biochemical and biophysical processes integrate to generate force and, ultimately, to regulate hemopoiesis into the bone marrow-matrix environment. To address this hypothesis, in this work we focus on the regulation of MK development by type I collagen. By atomic force microscopy analysis, we demonstrate that the tensile strength of fibrils in type I collagen structure is a fundamental requirement to regulate cytoskeleton contractility of human MKs through the activation of integrin-␣2␤1-dependent Rho-ROCK pathway and MLC-2 phosphorylation. Most importantly, this mechanism seemed to mediate MK migration, fibronectin assembly, and platelet formation. On the contrary, a decrease in mechanical tension caused by N-acetylation of lysine side chains in type I collagen completely reverted these processes by preventing fibrillogenesis. (Blood. 2011;118(16):4449-4453) IntroductionCollagen fibrils are the main architectural element in several tissues and bones, and the self-assembly processes involved in collagen fibrillogenesis are of enormous importance to matrix pathology and proper cell development. 1 Type I collagen plays a critical role in the bone marrow niche environment, preventing platelet formation by MKs through the activation of integrin-␣2␤1 and the Rho-ROCK pathway. 2,3,4 Changes in mechanical properties are tightly coupled in vivo to changes in matrix composition, crosslinking, and density. 5 Moreover, different signaling pathways, related to the transduction of mechanical signals into biochemical responses, regulate cell behavior. 6 However, the question on how biochemical and biophysical processes integrate to generate force and, ultimately, to regulate MK fate, is still open. Therefore, our purpose for the present work was to focus on MK function related to type I collagen organization and biomechanics. MethodsType I collagen from bovine tendon was modified as previously described 9 and as reported in supplemental Methods (available on the Blood Web site; see the Supplemental Materials link at the top of the online article). Antibodies and solid-phase binding assay are described in supplemental Methods. MKs were differentiated from human cord blood progenitors as previously described as well as proplatelet and spreading assay, electron and immunofluorescence microscopy, second harmonic generation, and Western blotting analysis. 4,7,8 Human cord blood was collected on informed consent of the parents, with approval from the ethical committee of the Istituto di Ricovero e Cura a Carattere Scientifico Policlinico San Matteo Foundation in accordance with the principles of the Declaration of Helsinki. Binding of cells to different collagen preparations was evaluated with 25 ϫ 10 3 cells seeded for 1 hour in Stem Span medium (Stem Cell Technologies) in a 96-well plate precoated with 5 g of each collagen prepa...

The importance of calcium in the regulation of megakaryocyte function

“…3,4,34 Importantly, it is known that different autocrine loops are fundamental modulators of Mk development. 5,6 With regard to this, we demonstrated that ADP is constitutively released by human Mks in vitro and that it is a crucial molecule in the regulation of platelet production.…”

“…the serine/threonine-specific kinase Akt, the mitogen-activated protein kinases ERK, the tyrosine kinases FAK, Src Syk and the myosin light chain, MLC). 7,[32][33][34][35] We showed that, upon stimulation with ADP (25 μM), the extent of phosphorylation of all tested molecules increased in adherent Mks on both fibrinogen and fibronectin substrates ( Figure 5A). We next focused our attention exclusively on the role of Ca 2+ on ADP signaling.…”

“…36 Importantly, we demonstrated that only type I collagen is able to inhibit proplatelet formation by sustained phosphorylation of MLC in human Mks. 34 Thus we decided to test the role of SOCE on Mk behavior on this ECM component. Interestingly, treatment with BTP-2 (20 μM) significantly inhibited Mk adhesion with a decreased number of cells displaying cytoskeleton reorganization with respect to non-treated controls ( Figure 7A-C).…”

Label free monitoring of megakaryocytic development and proplatelet formation in vitro

“…2(D)), caused by the endomitotic processes reaching maximal rate. On Day 1, as fibronectin sustains MK terminal differentiation [25], a significant increase in mature MKs is observed (stages IV-VI) (Fig. 2(A)), with their respective nuclear contribution to the cellular volume substantially decreasing, as cytoplasmic expansion and proplatelet extension dominate in these later phases (Fig.…”

Differential β3Integrin Expression Regulates the Response of Human Lung and Cardiac Fibroblasts to Extracellular Matrix and Its Components

“…50 Thus, the difference in size and orientation of collagen fibrils between collagen coatings and intact ECM may result in different substrate nanomechanics. Collagen nanomechanical properties have been shown to affect contractility and spreading of human megakaryocytes, 51 but it is unclear what impact these properties may have on fibroblast function.…”