Hyaluronic acid synthesis, degradation, and crosslinking in equine osteoarthritis: TNF-α-TSG-6-mediated HC-HA formation

Background TNF-α-stimulated gene 6 (TSG-6) protein, a TNF-α-responsive hyaladherin, possesses enzymatic activity that can catalyze covalent crosslinks of the polysaccharide hyaluronic acid (HA) to another protein to form heavy chain-hyaluronic acid (HC-HA) complexes in pathological conditions such as osteoarthritis (OA). Here, we examined HA synthase and inflammatory gene expression; synovial fluid HA, TNF-α, and viscosity; and TSG-6-mediated HC-HA complex formation in an equine OA model. The objectives of this study were to (1) evaluate the TNF-α-TSG-6-HC-HA signaling pathway across multiple joint tissues, including synovial membrane, cartilage, and synovial fluid, and (2) determine the impact of OA on synovial fluid composition and biophysical properties. Methods HA and inflammatory cytokine concentrations (TNF-α, IL-1β, CCL2, 3, 5, and 11) were analyzed in synovial fluid from 63 OA and 25 control joints, and HA synthase (HAS1-3), TSG-6, and hyaluronan-degrading enzyme (HYAL2, HEXA) gene expression was measured in synovial membrane and cartilage. HA molecular weight (MW) distributions were determined using agarose gel electrophoresis and solid-state nanopore measurements, and HC-HA complex formation was detected via immunoblotting and immunofluorescence. SEC-MALS was used to evaluate TSG-6-mediated HA crosslinking, and synovial fluid and HA solution viscosities were analyzed using multiple particle-tracking microrheology and microfluidic measurements, respectively. Results TNF-α concentrations were greater in OA synovial fluid, and TSG6 expression was upregulated in OA synovial membrane and cartilage. TSG-6-mediated HC-HA complex formation was greater in OA synovial fluid and tissues than controls, and HC-HA was localized to both synovial membrane and superficial zone chondrocytes in OA joints. SEC-MALS demonstrated macromolecular aggregation of low MW HA in the presence of TSG-6 and inter-α-inhibitor with concurrent increases in viscosity. Conclusions Synovial fluid TNF-α concentrations, synovial membrane and cartilage TSG6 gene expression, and HC-HA complex formation were increased in equine OA. Despite the ability of TSG-6 to induce macromolecular aggregation of low MW HA with resultant increases in the viscosity of low MW HA solutions in vitro, HA concentration was the primary determinant of synovial fluid viscosity rather than HA MW or HC-HA crosslinking. The TNF-α-TSG-6-HC-HA pathway may represent a potential therapeutic target in OA

A Multiwell Platform for Studying Stiffness-Dependent Cell Biology

Adherent cells are typically cultured on rigid substrates that are orders of magnitude stiffer than their tissue of origin. Here, we describe a method to rapidly fabricate 96 and 384 well platforms for routine screening of cells in tissue-relevant stiffness contexts. Briefly, polyacrylamide (PA) hydrogels are cast in glass-bottom plates, functionalized with collagen, and sterilized for cell culture. The Young's modulus of each substrate can be specified from 0.3 to 55 kPa, with collagen surface density held constant over the stiffness range. Using automated fluorescence microscopy, we captured the morphological variations of 7 cell types cultured across a physiological range of stiffness within a 384 well plate. We performed assays of cell number, proliferation, and apoptosis in 96 wells and resolved distinct profiles of cell growth as a function of stiffness among primary and immortalized cell lines. We found that the stiffness-dependent growth of normal human lung fibroblasts is largely invariant with collagen density, and that differences in their accumulation are amplified by increasing serum concentration. Further, we performed a screen of 18 bioactive small molecules and identified compounds with enhanced or reduced effects on soft versus rigid substrates, including blebbistatin, which abolished the suppression of lung fibroblast growth at 1 kPa. The ability to deploy PA gels in multiwell plates for high throughput analysis of cells in tissue-relevant environments opens new opportunities for the discovery of cellular responses that operate in specific stiffness regimes

Integrin Clustering Is Driven by Mechanical Resistance from the Glycocalyx and the Substrate

Integrins have emerged as key sensory molecules that translate chemical and physical cues from the extracellular matrix (ECM) into biochemical signals that regulate cell behavior. Integrins function by clustering into adhesion plaques, but the molecular mechanisms that drive integrin clustering in response to interaction with the ECM remain unclear. To explore how deformations in the cell-ECM interface influence integrin clustering, we developed a spatial-temporal simulation that integrates the micro-mechanics of the cell, glycocalyx, and ECM with a simple chemical model of integrin activation and ligand interaction. Due to mechanical coupling, we find that integrin-ligand interactions are highly cooperative, and this cooperativity is sufficient to drive integrin clustering even in the absence of cytoskeletal crosslinking or homotypic integrin-integrin interactions. The glycocalyx largely mediates this cooperativity and hence may be a key regulator of integrin function. Remarkably, integrin clustering in the model is naturally responsive to the chemical and physical properties of the ECM, including ligand density, matrix rigidity, and the chemical affinity of ligand for receptor. Consistent with experimental observations, we find that integrin clustering is robust on rigid substrates with high ligand density, but is impaired on substrates that are highly compliant or have low ligand density. We thus demonstrate how integrins themselves could function as sensory molecules that begin sensing matrix properties even before large multi-molecular adhesion complexes are assembled

The normal breast microenvironment of premenopausal women differentially influences the behavior of breast cancer cells in vitro and in vivo

<p>Abstract</p> <p>Background</p> <p>Breast cancer studies frequently focus on the role of the tumor microenvironment in the promotion of cancer; however, the influence of the normal breast microenvironment on cancer cells remains relatively unknown. To investigate the role of the normal breast microenvironment on breast cancer cell tumorigenicity, we examined whether extracellular matrix molecules (ECM) derived from premenopausal African-American (AA) or Caucasian-American (CAU) breast tissue would affect the tumorigenicity of cancer cells <it>in vitro </it>and <it>in vivo</it>. We chose these two populations because of the well documented predisposition of AA women to develop aggressive, highly metastatic breast cancer compared to CAU women.</p> <p>Methods</p> <p>The effects of primary breast fibroblasts on tumorigenicity were analyzed via real-time PCR arrays and mouse xenograft models. Whole breast ECM was isolated, analyzed via zymography, and its effects on breast cancer cell aggressiveness were tested <it>in vitro </it>via soft agar and invasion assays, and <it>in vivo </it>via xenograft models. Breast ECM and hormone metabolites were analyzed via mass spectrometry.</p> <p>Results</p> <p>Mouse mammary glands humanized with premenopausal CAU fibroblasts and injected with primary breast cancer cells developed significantly larger tumors compared to AA humanized glands. Examination of 164 ECM molecules and cytokines from CAU-derived fibroblasts demonstrated a differentially regulated set of ECM proteins and increased cytokine expression. Whole breast ECM was isolated; invasion and soft agar assays demonstrated that estrogen receptor (ER)^-, progesterone receptor (PR)/PR^-cells were significantly more aggressive when in contact with AA ECM, as were ER⁺/PR⁺cells with CAU ECM. Using zymography, protease activity was comparatively upregulated in CAU ECM. In xenograft models, CAU ECM significantly increased the tumorigenicity of ER⁺/PR⁺cells and enhanced metastases. Mass spectrometry analysis of ECM proteins showed that only 1,759 of approximately 8,000 identified were in common. In the AA dataset, proteins associated with breast cancer were primarily related to tumorigenesis/neoplasia, while CAU unique proteins were involved with growth/metastasis. Using a novel mass spectrometry method, 17 biologically active hormones were measured; estradiol, estriol and 2-methoxyestrone were significantly higher in CAU breast tissue.</p> <p>Conclusions</p> <p>This study details normal premenopausal breast tissue composition, delineates potential mechanisms for breast cancer development, and provides data for further investigation into the role of the microenvironment in cancer disparities.</p

Do Femtonewton Forces Affect Genetic Function? A Review

Protein-Mediated DNA looping is intricately related to gene expression. Therefore any mechanical constraint that disrupts loop formation can play a significant role in gene regulation. Polymer physics models predict that less than a piconewton of force may be sufficient to prevent the formation of DNA loops. Thus, it appears that tension can act as a molecular switch that controls the much larger forces associated with the processive motion of RNA polymerase. Since RNAP can exert forces over 20 pN before it stalls, a ‘substrate tension switch’ could offer a force advantage of two orders of magnitude. Evidence for such a mechanism is seen in recent in vitro micromanipulation experiments. In this article we provide new perspective on existing theory and experimental data on DNA looping in vitro and in vivo . We elaborate on the connection between tension and a variety of other intracellular mechanical constraints including sequence specific curvature and supercoiling. In the process, we emphasize that the richness and versatility of DNA mechanics opens up a whole new paradigm of gene regulation to explore.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41816/1/10867_2005_Article_9002.pd

Integrins sense extracellular-matrix rigidity to regulate mammary-epithelial tissue phenotype

Tumors are stiffer than normal tissue, but whether this biophysical alteration actively contributes to malignant behavior has not been assessed. Cellular integrins physically interact with the tissue environment by tugging on the extracellular matrix (ECM). Because integrins are mechanotransducers that regulate cell fate, we asked if tissue stiffness could regulate mammary epithelial tissue behavior by modulating integrin adhesion assembly. We found that compliant matrices equivalent in stiffness to the normal mammary gland induce the assembly of focal complexes that supported normal morphogenesis. Rigid matrices, on the other hand, drove focal adhesion maturation to disrupt tissue morphogenesis and enhance epithelial growth. We determined that the focal complex to focal adhesion transition is triggered by integrin clustering. To better understand how matrix stiffness influences integrin clustering, we built a stochastic model of integrin-ligand interactions within a deformable cell-ECM interface. With this model, we showed that integrins spontaneously cluster on rigid matrices through a glycocalyx-mediated mechanism that does not require cytoskeletal adaptors or cell contractility. Increasing matrix compliance in the model, however, diminished integrin clustering. Experimentally, we determined that clustering ultimately was linked to focal adhesion maturation by a tension-dependent feedback circuit. Specifically, integrin clustering stimulated ERK and Rho activation, which in turn increased cytoskeletal contractility to drive focal adhesion assembly and additional integrin clustering. Matrix rigidity was thus sensed, at least in part, by a myosin-independent, integrin-clustering mechanism and a myosin-dependent, cell-signaling circuit. Perturbation of any aspect of this mechano-sensory process compromised tissue phenotype. Indeed, enhancing matrix rigidity, inducing integrin clustering, or increasing ERK- or Rho-mediated tension disrupted epithelial morphogenesis in 3D culture models. Remarkably, we found that highly-contractile, EGF-transformed epithelia with high ERK and Rho activity could be phenotypically reverted to tissues lacking focal adhesions if Rho-contractility or EGF was decreased. These studies suggest new possibilities for cancer intervention based on normalizing the mechano-biology of the tumor

Integrin clustering is driven by mechanical resistance from the glycocalyx and the substrate.

Integrins have emerged as key sensory molecules that translate chemical and physical cues from the extracellular matrix (ECM) into biochemical signals that regulate cell behavior. Integrins function by clustering into adhesion plaques, but the molecular mechanisms that drive integrin clustering in response to interaction with the ECM remain unclear. To explore how deformations in the cell-ECM interface influence integrin clustering, we developed a spatial-temporal simulation that integrates the micro-mechanics of the cell, glycocalyx, and ECM with a simple chemical model of integrin activation and ligand interaction. Due to mechanical coupling, we find that integrin-ligand interactions are highly cooperative, and this cooperativity is sufficient to drive integrin clustering even in the absence of cytoskeletal crosslinking or homotypic integrin-integrin interactions. The glycocalyx largely mediates this cooperativity and hence may be a key regulator of integrin function. Remarkably, integrin clustering in the model is naturally responsive to the chemical and physical properties of the ECM, including ligand density, matrix rigidity, and the chemical affinity of ligand for receptor. Consistent with experimental observations, we find that integrin clustering is robust on rigid substrates with high ligand density, but is impaired on substrates that are highly compliant or have low ligand density. We thus demonstrate how integrins themselves could function as sensory molecules that begin sensing matrix properties even before large multi-molecular adhesion complexes are assembled

Integrin clustering is driven by mechanical resistance from the glycocalyx and the substrate.

Integrins have emerged as key sensory molecules that translate chemical and physical cues from the extracellular matrix (ECM) into biochemical signals that regulate cell behavior. Integrins function by clustering into adhesion plaques, but the molecular mechanisms that drive integrin clustering in response to interaction with the ECM remain unclear. To explore how deformations in the cell-ECM interface influence integrin clustering, we developed a spatial-temporal simulation that integrates the micro-mechanics of the cell, glycocalyx, and ECM with a simple chemical model of integrin activation and ligand interaction. Due to mechanical coupling, we find that integrin-ligand interactions are highly cooperative, and this cooperativity is sufficient to drive integrin clustering even in the absence of cytoskeletal crosslinking or homotypic integrin-integrin interactions. The glycocalyx largely mediates this cooperativity and hence may be a key regulator of integrin function. Remarkably, integrin clustering in the model is naturally responsive to the chemical and physical properties of the ECM, including ligand density, matrix rigidity, and the chemical affinity of ligand for receptor. Consistent with experimental observations, we find that integrin clustering is robust on rigid substrates with high ligand density, but is impaired on substrates that are highly compliant or have low ligand density. We thus demonstrate how integrins themselves could function as sensory molecules that begin sensing matrix properties even before large multi-molecular adhesion complexes are assembled