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Soft Hyaluronic Gels Promote Cell Spreading, Stress Fibers, Focal Adhesion, and Membrane Tension by Phosphoinositide Signaling, not Traction Force Kalpana Mandal, Dikla Raz-Ben Aroush, Zachary Tobias Graber, Bin Wu, Chan Young Park, Jeffrey J. Fredberg, Wei Guo, Tobias Baumgart, and Paul A. Janmey ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b05286 • Publication Date (Web): 30 Nov 2018 Downloaded from http://pubs.acs.org on December 1, 2018
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Soft Hyaluronic Gels Promote Cell Spreading, Stress Fibers, Focal Adhesion, and Membrane Tension by Phosphoinositide Signaling, not Traction Force Kalpana Mandal1 *, Dikla Raz-Ben Aroush1,2, Zachary Tobias Graber3, Bin Wu4, Chan Young Park5, Jeffery J. Fredberg5, Wei Guo4, Tobias Baumgart3, Paul A. Janmey1 * 1Institute
for Medicine and Engineering, Department of Physiology, University of Pennsylvania, Philadelphia, PA 19104 USA,
2Kennedy
Institute of Rheumatology, University of Oxford, Oxford OX3 7FY, UK, 3Department of Chemistry University of
Pennsylvania, Philadelphia, PA 19104 USA, 4Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA 5School
of Public Health, Harvard University, Boston, MA 02115, USA.
* Corresponding
authors:
[email protected],
[email protected] Abstract: Cells respond to both physical and chemical aspects of their substrate. Whether intracellular signals initiated by physical stimuli are fundamentally different from those elicited by chemical stimuli is an open question. Here we show that the requirement for a stiff substrate and therefore high cellular tension in order for cells to produce large focal adhesions and stress fibers is obviated when a soft substrate contains both hyaluronic acid (HA) and an integrin ligand (collagen I). HA is a major ECM component that is often upregulated during wound healing and tumor growth. HA together with collagen I promote hepatocellular carcinoma cell (Huh7) spreading on very soft substrates (300 Pa), resulting in morphology and motility similar to what these cells develop only on stiff substrates (>30 kPa) formed by polyacrylamide that contain collagen but no HA. The effect of HA requires turnover of polyphosphoinositides and leads to activation of AKT. Inhibition of polyphosphoinositide turnover causes Huh7 cells and fibroblasts to decrease spreading and detach, whereas cells on stiffer substrates show almost no response. Traction force microscopy shows that the cell maintains a low strain energy and net contractile moment on HA substrates as compared to stiff polyacrylamide substrates. Membrane tension measured by tether pulling is similar on soft HA and stiff polyacrylamide substrates. These results suggest that simultaneous signaling stimulated by HA and an integrin ligand can generate phosphoinositide-mediated signals to the cytoskeleton that reproduce those generated by high cellular tension. Keywords: hyaluronic acid, PIP3, PIP2, HCC, TFM, membrane tension Many cell types alter their structure and function depending on the mechanical properties of the materials to which they adhere and on the type of adhesion receptor by which they bind.1–4 In vivo, cells engage their
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extracellular matrix (ECM) both by mechanosensitive adhesion complexes and by other surface receptors for ECM components that cannot act as adhesive anchors, but that potentially modify the mechanical signals transduced at the cell/ECM interface. Cellular reaction to extracellular matrix (ECM) depends upon the specificity of the ligand binding.2,5 Previous studies found that cardiomyocytes grown on soft hyaluronic acid gels coated with fibronectin (HA-Fn) developed well-structured sarcomeres despite the very low elastic modulus of this material, suggesting that the role of the cardiac jelly in early stages of cardiogenesis is more than that of a passive coupling matrix between the myocardial-cardiomyocytes and endocardialendothelial cell layers enveloping it.6,7 Hyaluronic acid (or hyaluronan (HA)), a non-sulfated glycosaminoglycan polysaccharide, is a major ECM component that plays an important role in development, wound healing, cancer progression, and organizes the supramolecular assembly of proteoglycans.
8–10
In normal tissue, HA amount is determined by the
balance between HA synthesis and degradation. An over production of HA or upregulation of HA receptors facilitates cell migration, invasion of tumor cells or rapid tumor growth.11 Breast cancer cells synthesize more HA than normal cells, and high HA production is correlated with poor patient survival in various types of cancer including prostate, breast and ovarian cancer.12,13 HA localizes at the leading edge of the tumor.14 HA is also upregulated in hepatocellular carcinoma, suggesting a pathophysiologic relevance to investigating how Huh7 cells respond to HA in this context. Cells on HA substrates of stiffness 300 Pa can spread as much as on very stiff substrates (10 kPa) when both are coated with a ligand for integrins, usually either fibronectin or collagen I.15–17 Cell interaction with ECM-bound HA is mediated by CD44, CD168 (RHAMM), and other cell surface receptors. CD44 is overexpressed in many cancers and has been shown to promote angiogenesis from tumors.18,19 Cell interaction with ECM-HA through these receptors regulates many cell signaling pathways including RhoGTPase, transforming growth factor beta (TGF- β) and focal adhesion kinase (FAK) mediated pathways.20 Prior studies show a large effect of HA on cell morphology, stiffness, and contractility on soft gels containing Fn, but the molecular mechanism by which HA in the matrix exerts this effect is unexplored. This study is designed to determine if signals downstream of phosphatidylinositol-4,5-bisphosphate (PIP2) or phosphatidylinositol-3,4,5-trisphosphate (PIP3) control the effect of HA by inhibiting phosphatidylinositol-3kinase (PI3K) using wortmannin and LY296004 or sequestering PIP2 by the peptide PBP10. Previous work showed that a cell-permeant fluorescent peptide derivative based on the PIP2-binding site of gelsolin can reversibly destabilize the actin filament network of fibroblasts and other cell types bound to glass, but does not alter their spread area or detach them from the substrate. 21 Manipulation of enzymes involved in PIP2
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production showed that increasing cellular PIP2 levels massively increases actin assembly and stress fiber formation, whereas increasing PIP2 degradation destabilizes actin assembly.22–25 PIP3, which is the lipid product of PI3K, regulates cell migration, proliferation, and growth.26 A prominent effector of PIP3 is the serine-threonine kinase Akt, which binds to the membrane via PIP3, facilitating its activation by phosphoinositide-dependent kinase 1 (PDK1). In addition, PIP3 is amplified in some regions of the membrane and initiates a signaling wave which in turn remodels actin structures along the perimeter of the propagating PIP3 wave.27,28 Here we investigate the role of HA in PIP2 and PIP3 mediated signaling in hepatocellular carcinoma cells and fibroblasts. We explore the cell-substrate interaction process in HA-mediated adhesion as well as on stiff polyacrylamide (PAA) substrates by traction force microscopy and by measuring the plasma membrane tension, which depends on contributions from lipid bilayer surface tension, hydrostatic pressure across the membrane, and the interaction between the plasma membrane with the underlying actincytoskeleton mediated by contractility of the actomyosin cortex.24,29–32 The ratio of PIP2/PIP3 coordinates actomyosin contractility by limiting myosin recruitment during plasma membrane expansion in tissue morphogenesis, thus ensuring proper cell shape.33 Results/Discussion Cell spreading on HA and 30 kPa substrate: To study cell spreading and intracellular organization, Huh7 cells were cultured on 300 Pa HA- collagen, 300 Pa HA-FN, and 30 kPa or 500 Pa collagen coated PAA substrates. Phase contrast images were taken after 24 hrs of plating, and cell areas were measured. The result shows that the average cell area is significantly lower on 500 Pa -PAA compared to 30 kPa - PAA or 300 Pa HA substrates (Fig 1A). There was no significant area difference between cells on 30 kPa PAA collagen and 300 Pa HA coated either with fibronectin or collagen (Fig 1A). This result confirms the previous finding that cells on soft HA substrates spread as much as they do on stiff substrates without HA 15. The extent of spreading depends on the density of collagen on the gel surface, and this dependence saturates when the concentration of collagen in the coating medium exceeds 60 µg/ml (Fig S1A,B). One of the most striking features of cells grown on soft HA gels are actin bundles (Fig 1) and prominent focal adhesions (Fig 1B) that appear on substrates that are much too soft to support these structures if the substrate is 500 Pa PAA. Immunostaining images shows that the cells are able to form organized microtubule and vimentin networks on both of these two substrates but not on soft PAA substrates (Fig 1B).
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Figure1: Cell Spreading on HA and PAA substrate. Huh7 cell spreading on different substrate with varying stiffness and ligand binding (Collagen (Col) and Fibronectin (FN)). Cells are grown on HA and 30kPa substrates for 24 hrs (A) Phase contrast image of the cells on 30 kPa-Col, 30 kPaFN, HA-FN, HA-col, 500 Pa Col substrates are shown. (Scale bar, 20 μm). (right) Huh7 cell area quantified on HA and PAA gel. Error bars represent standard error of the mean of N30kPa-col =34, N30kPa-FN =29 and NHA-FN=25, NHA-col =36, N500Pa-col =26) cells on these substrates respectively. p-values are determined from Student’s t-test for unpaired samples f (** p
