Augmentation of integrin-mediated mechanotransduction by hyaluronic acid

Abstract

Changes in tissue and organ stiffness occur during development and are frequently symptoms of disease. Many cell types respond to the stiffness of substrates and neighboring cells in vitro and most cell types increase adherent area on stiffer substrates that are coated with ligands for integrins or cadherins. In vivo cells engage their extracellular matrix (ECM) by multiple mechanosensitive adhesion complexes and other surface receptors that potentially modify the mechanical signals transduced at the cell/ECM interface. Here we show that hyaluronic acid (also called hyaluronan or HA), a soft polymeric glycosaminoglycan matrix component prominent in embryonic tissue and upregulated during multiple pathologic states, augments or overrides mechanical signaling by some classes of integrins to produce a cellular phenotype otherwise observed only on very rigid substrates. The spread morphology of cells on soft HA-fibronectin coated substrates, characterized by formation of large actin bundles resembling stress fibers and large focal adhesions resembles that of cells on rigid substrates, but is activated by different signals and does not require or cause activation of the transcriptional regulator YAP. The fact that HA production is tightly regulated during development and injury and frequently upregulated in cancers characterized by uncontrolled growth and cell movement suggests that the interaction of signaling between HA receptors and specific integrins might be an important element in mechanical control of development and homeostasis.

Keywords: Cell spreading; Extracellular matrix; Hyaluronic acid; Mechanosensing; Traction stresses; Yes associated protein (YAP).

Figure 1. Development of stress fibers and focal adhesions on soft HA-Fn gels

Human mesenchymal stem cells (hMSCs), neonatal rat ventricular myocytes and fibroblasts, NIH 3T3 fibroblasts, and human umbilical vein endothelial cells (HUVECS) were plated on 300 Pa HA-fibronectin gels, 300 Pa PAA-fibronectin gels, or Fn-coated glass. Actin is stained with rhodamine-phalloidin (red) and cell nuclei are stained by DAPI (blue). Alpha actinin is stained green for myocytes, and paxillin is shown in white for endothelial cells. Scale bar represents 20μm.

Figure 2. Enhanced cell spreading response on HA-fibronectin hydrogels

(A) Myocyte spread area on HA and PAA gels with identical shear modulus coated with varying amounts of Fn (n >100 cells). (B) MSC spread area on varying stiffnesses of polyacrylamide or HA gels coated with saturating amounts of Fn (n>100 cells). (C) Quantification of early fibroblast cell spread area over time on varying substrates (n>3). Error bars ±1 S.E..

Figure 3. Cell spreading response as a function of ligand type

(A) Mesenchymal stem cell (MSC) spread area on different stiffness polyacrylamide or HA gels coated with saturating amounts of Fn, Collagen I, Poly-l-lysine (PLL), Laminin and N-cadherin. (B) Schematic representation of the sandwich construct, showing cells adhered to 300Pa PAA-Fn gels at the bottom with different HA-ligand compositions at the top. (C) MSC spread areas for cells sandwiched between 300Pa PAA-Fn and HA-alone/Fn/Col-I. *p<0.05, error bars ±1 SEM for n= 50 cells.

Figure 4. Cell spreading in response to HA incorporated with RGD alone or RGD containing ligands

(A) Cardiac fibroblasts and myocytes plated on HA-Fn and HA-RGD show comparable spread area, stress-fiber formation (f-actin; green) and myofibrillar assembly (α-actinin; green). (B) Spread area magnitudes of myocytes plated on fibronectin, RGD, collagen IV (Col IV) and I (Col I). *p<0.01, error bars ±1 SEM for n>100 cells.

Figure 5. Enhanced cells proliferation on 300Pa HA-Fn gels

Proliferation rates of MSCs (A), fibroblasts (B) and HUVECs (C) on HA with or without different integrin ligands, and comparison of growth rates on PAA-Fn gels or tissue culture plastic (TCP). Error bars ±1 SEM for n>5 fields.

Figure 6. Cell and hydrogel material properties measured by atomic force microscopy

(A) Storage modulus of HA gels with different ECM ligands. (B, C) Comparison of cell stiffness and cell area on Fn coated HA and glass. (D) Left: image of a 3T3 fibroblast spread on an HA-FN gel prepared on top of a CELLocate coverslip. AFM was used to indent three areas of the cell (red dots) and three areas of the HA-Fn gel within 30 μm of the cell edge (blue dots). Right: HA-Fn gel shown in left image after cell has been detached. Blue dots indicate areas of the gel to which the cell had been previously attached that were indented by AFM. (E) Graph shows the measured elastic modulus of 3T3 fibroblasts attached to an HA-Fn gel, the HA-Fn gel in proximity of the attached cell and the area of the HA-Fn gel beneath the cell after cell detachment. No significant difference was observed between each set of measurements. *p<0.01, error bars ±1 SEM for n>6 cells; scale bar represents 75μm.

Figure 7. Cell traction forces exerted on 2D and within 3D hyaluronan gels

(A) Mouse embryonic fibroblasts were cultured on fibronectin micro-patterned dot grid (1μm diameter) hyaluronan substrates. Traction stresses were estimated using the measured displacement vectors of the Fn dot (n=3 cells). (B) Resting traction stresses of cardiac myocytes calculated from the displacement of fluorescent beads incorporated in 300 Pa HA-Fn or PAA-Fn gels. *p<0.05 error bars ±1 SEM for n>7 cells, scale bar indicates 20μm. (C) cardiac cell mediated gel compaction n=10, (D) measured gel stiffness E (Young’s modulus for n=4) and (E) average area of myocytes with the compacted gels n=50, of constructs formed by embedding cardiac myocytes and fibroblasts (10⁶/ml) within gels composed of 1 mg/mL fibrin and 0.25 mg/mL collagen after 1 week. Error bars ±1 S.D.

Figure 8. Measurement of YAP localization as a function of stiffness on PAA-fn and HA-Fn hydrogels

(A, B) Nuclear localization of Yap in fibroblasts with increasing stiffness on PAA-Fn, but not on HA-Fn (n= 250 cells from 3-different preparations).