Objectives-To investigate the role of matrix metalloproteinase (MMP)-13/collagenase-3 in osteoarthritis (OA).Methods-Surgically-induced OA in knees of MMP-13 knock out (KO) and wild type (WT) mice was compared. Femoral and tibial cartilage aggrecan loss (0-3), erosion (0-7) and chondrocyte hypertrophy (0-1), as well as osteophyte size (0-3) and maturity (0-3) were histologically scored. Serial sections were stained for collagen type X and the MMP-generated aggrecan neo-epitope DIPEN.Results-Following surgery, aggrecan loss and cartilage erosion were more severe in the tibia than femur (p<0.01) and tibial cartilage erosion increased with time (p<0.05) in WT mice. Cartilaginous osteophytes were present at 4 weeks and underwent ossification, with size and maturity increasing by 8 weeks (p<0.01). There was no difference between genotypes in aggrecan loss or cartilage erosion at 4 weeks. Tibial cartilage erosion in KO mice was less than WT at 8 weeks (p<0.02). Cartilaginous osteophytes were larger in KO at 4 weeks (p<0.01), but by 8 weeks osteophyte maturity and size were no different from WT. Articular chondrocyte hypertrophy with positive type X collagen and DIPEN staining occurred in both WT and KO joints.Conclusions-These studies have confirmed that structural cartilage damage in mouse experimental OA is dependent on MMP-13 activity. Chondrocyte hypertrophy is not regulated by MMP-13 activity in this model and does not in itself lead to cartilage erosion. MMP-13 deficiency can inhibit cartilage erosion in the presence of aggrecan depletion, supporting the potential for therapeutic intervention in established OA with MMP-13 inhibitors.Progressive erosion of articular cartilage is a significant determinant of prognosis and the need for joint replacement surgery in osteoarthritis (OA). Proteolysis of the principal NIH Public Access Author ManuscriptArthritis Rheum. Author manuscript; available in PMC 2010 December 1. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript cartilage extracellular matrix constituents, aggrecan and the type II/IX/XI collagen network, directly causes erosion as well as predisposing the tissue to mechanical disruption even with loading at physiological levels. Aggrecan proteolysis and loss precedes and may be prerequisite for subsequent collagenolysis (1). A distintegrin and metalloproteinase with thrombospondin repeat (ADAMTS) enzymes are responsible for pathological aggrecanolysis (2,3). ADAMTS-5 is the predominant arthritis-associated enzyme in mice, since animals deficient in ADAMTS-5 activity are protected from cartilage erosion in OA and inflammatory arthritis (4-6). Ablating the ADAMTS cleavage site in the interglobular domain of aggrecan also blocks cartilage structural damage, confirming that the effect in ADAMTS-5-deficient mice is due to inhibition of aggrecanolysis (7).The above studies demonstrate that inhibiting the initiation of aggrecan loss can prevent subsequent structural cartilage damage/erosion in arthritis. Clinically, it is likely that e...
Cancer invasion through dense extracellular matrices (ECMs) is mediated by matrix metalloproteinases (MMPs) which degrade the ECM thereby creating paths for migration. However, how this degradation influences the phenotype of cancer cells is not fully clear. Here we address this question by probing the function of MMPs in regulating biophysical properties of cancer cells relevant to invasion. We show that MMP catalytic activity regulates cell spreading, motility, contractility and cortical stiffness by stabilizing integrins at the membrane and activating focal adhesion kinase. Interestingly, cell rounding and cell softening on stiff gels induced by MMP inhibition is attenuated on MMP pre-conditioned surfaces. Together, our results suggest that MMP catalytic activity regulates invasiveness of cancer cells by modulating integrins.
Fabrication of nanoscale DNA devices to generate 3D nano-objects with precise control of shape, size, and presentation of ligands has shown tremendous potential for therapeutic applications. The interactions between the cell membrane and different topologies of 3D DNA nanostructures are crucial for designing efficient tools for interfacing DNA devices with biological systems. The practical applications of these DNA nanocages are still limited in cellular and biological systems owing to the limited understanding of their interaction with the cell membrane and endocytic pathway. The correlation between the geometry of DNA nanostructures and their internalization efficiency remains elusive. We investigated the influence of the shape and size of 3D DNA nanostructures on their cellular internalization efficiency. We found that one particular geometry, i.e., the tetrahedral shape, is more favored over other designed geometries for their cellular uptake in 2D and 3D cell models. This is also replicable for cellular processes like cell invasion assays in a 3D spheroid model, and passing the epithelial barriers in in vivo zebrafish model systems. Our work provides detailed information for the rational design of DNA nanodevices for their upcoming biological and biomedical applications.
Invadopodia are micron-sized invasive structures that mediate extracellular matrix (ECM) degradation through a combination of membrane-bound and soluble matrix metalloproteinases (MMPs). However, how such localized degradation is converted into pores big enough for cancer cells to invade, and the relative contributions of membrane-bound versus soluble MMPs to this process remain unclear. In this article, we address these questions by combining experiments and simulations. We show that in MDA-MB-231 cells, an increase in ECM density enhances invadopodia-mediated ECM degradation and decreases inter-invadopodia spacing. ECM degradation is mostly mediated by soluble MMPs, which are activated by membrane-bound MT1-MMP. We present a computational model of invadopodia-mediated ECM degradation, which recapitulates the above observations and identifies MMP secretion rate as an important regulator of invadopodia stability. Simulations with multiple invadopodia suggest that inter-invadopodia spacing and MMP secretion rate collectively dictate the size of the degraded zones. Taken together, our results suggest that for creating pores conducive for cancer invasion, cells must tune inter-invadopodia spacing and MMP secretion rate in an ECM density-dependent manner, thereby striking a balance between invadopodia penetration and ECM degradation.
Acquired radioresistance accompanied with increased metastatic potential is a major hurdle in effective radiotherapy of breast cancers. However, the nature of their inter-dependence and the underlying mechanism remains largely intangible. By employing radioresistant (RR) cell lines, we herein demonstrate that MCF-7 RR cells display phenotypic and molecular alterations evocative of epithelial to mesenchymal transition (EMT) with increased traction forces and membrane ruffling culminating in boosted invasiveness. We then show that these changes can be attributed to overexpression of alpha-actinin-4 (ACTN4), with ACTN4 knockdown near-completely abrogating both radioresistance and EMT-associated changes. We further found that in MCF-7 RR cells, ACTN4 mediates the observed effects by activating AKT, and downstream AKT/GSK3β signalling. Though ACTN4 plays a similar role in mediating radioresistance and invasiveness in MDA-MB-231 RR cells, co-immunoprecipitation studies reveal that these changes are effected through increased association with AKT and not by overexpression of AKT. Taken together, our study identifies ACTN4/AKT/GSK3β as a novel pathway regulating radioresistance coupled invasion which can be further explored to improve the radiotherapeutic gain.
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