Deletion of TNFAIP6 Gene in Human Keratinocytes Demonstrates a Role for TSG-6 to Retain Hyaluronan Inside Epidermis.

TSG-6 is a soluble protein secreted in the extracellular matrix by various cell types in response to inflammatory stimuli. TSG-6 interacts with extracellular matrix molecules, particularly hyaluronan (HA), and promotes cutaneous wound closure in mice. Between epidermal cells, the discrete extracellular matrix contains HA and a tiny amount of TSG-6. However, challenges imposed to keratinocytes in reconstructed human epidermis revealed strong induction of TSG-6 expression, after exposure to T helper type 2 cytokines to recapitulate the atopic dermatitis phenotype or after fungal infection that causes secretion of cytokines and antimicrobial peptides. After both types of challenge, enhanced release of TSG-6 happens simultaneously with increased HA production. TSG-6 deficiency in N/TERT keratinocytes was created by inactivating TNFAIP6 using CRISPR/Cas9. Some TSG-6 -/- keratinocytes analyzed through scratch assays tend to migrate more slowly but produce reconstructed human epidermis that exhibits normal morphology and differentiation. Few significant alterations were noticed by transcriptomic analysis. Nevertheless, reduced HA content in TSG-6 -/- reconstructed human epidermis was observed, along with enhanced HA release into the culture medium, and this phenotype was even more pronounced after the challenging conditions. Reintroduction of cells producing TSG-6 in reconstructed human epidermis reduced HA leakage. Our results show a role for TSG-6 in sequestering HA between epidermal cells in response to inflammation.

Molecular size-dependent specificity of hyaluronan on functional properties, morphology and matrix composition of mammary cancer cells.

High levels of hyaluronan (ΗΑ), a major extracellular matrix (ECM) glycosaminoglycan, have been correlated with poor clinical outcome in several malignancies, including breast cancer. The high and low molecular weight HΑ forms exert diverse biological functions. Depending on their molecular size, ΗΑ forms either promote or attenuate signaling cascades that regulate cancer progression. In order to evaluate the effects of different ΗΑ forms on breast cancer cells' behavior, ΗΑ fragments of defined molecular size were synthesized. Breast cancer cells of different estrogen receptor (ER) status - the low metastatic, ERα-positive MCF-7 epithelial cells and the highly aggressive, ERß-positive MDA-MB-231 mesenchymal cells - were evaluated following treatment with HA fragments. Scanning electron microscopy revealed that HA fragments critically affect the morphology of breast cancer cells in a molecular-size dependent mode. Moreover, the ΗΑ fragments affect cell functional properties, the expression of major ECM mediators and epithelial-to-mesenchymal transition (ΕΜΤ) markers. Notably, treatment with 200 kDa ΗΑ increased the expression levels of the epithelial marker Ε-cadherin and reduced the expression levels of HA synthase 2 and mesenchymal markers, like fibronectin and snail2/slug. These novel data suggest that the effects of HA in breast cancer cells depend on the molecular size and the ER status. An in-depth understanding on the mechanistic basis of these effects may contribute on the development of novel therapeutic strategies for the pharmacological targeting of aggressive breast cancer.