Jozef Bizik

Formation and activation of fibroblast spheroids depend on fibronectin-integrin interaction

Clustering of fibroblasts into spheroids induces a massive proinflammatory, proteolytic and growth-factor response, named nemosis, which promotes tumor cell invasiveness and differentiation of leukemia cells. We have now sought to investigate mechanisms leading to the formation of multicellular spheroids and subsequent activation of fibroblasts (nemosis). Cell lines either lacking fibronectin expression (FN-/-) or expressing FN with a mutated integrin-binding site (FNRGE/RGE) were unable to form compact spheroids. Furthermore, inhibition of FN synthesis by siRNA or functional inhibition of FN or its integrins impaired spheroid formation (alpha5, beta1) and quenched fibroblast activation (alphaV). The integrin ligand GRGDSP hexapeptide interfered with spheroid formation and induced activation of fibroblasts. Surprisingly, a 70 kDa FN fragment, which prevents deposition of FN matrix but does not interfere with FN-integrin interaction, prevented spheroid formation only marginally and did not block the activation. Our results present a new mechanism of fibroblast activation, which is initiated by interaction of FN with its integrin receptors.

Human skin transcriptome during superficial cutaneous wound healing.

Healing of the epidermis is a crucial process for maintaining the skins defense integrity and its resistance to environmental threats. Compromised wound healing renders the individual readily vulnerable to infections and loss of body homeostasis. To clarify the human response of reepithelialization, we biopsied split‐thickness skin graft donor site wounds immediately before and after harvesting, as well as during the healing process 3 and 7 days thereafter. In all, 25 biopsies from eight patients qualified for the study. All samples were analyzed by genome‐wide microarrays. Here, we identified the genes associated with normal skin reepithelialization over time and organized them by similarities according to their induction or suppression patterns during wound healing. Our results provide the first elaborate insight into the transcriptome during normal human epidermal wound healing. The data not only reveal novel genes associated with epidermal wound healing but also provide a fundamental basis for the translational interpretation of data acquired from experimental models.

REGULATION OF THE PERICELLULAR ACTIVATION OF PLASMINOGEN AND ITS ROLE IN TISSUE‐DESTRUCTIVE PROCESSES

While it is generally recognized today that cells use specific adhesion proteins such as fibronectin and laminin, both in cell adhesion and migration, it is equally clear that pericelldar proteolysis is critically involved in the invasion of malignant and certain normal cells through the extracellular matrices, both interstitial connective tissue matrices and basement membranes. There is interesting new evidence suggesting that a key component in pericellular proteolysis, i.e. the urokinase-receptor complex, may have a role also in both cell adhesion and cell migration. In addition there is direct evidence that adhesion proteins of pericellular matrix can provide a molecular link between the membrane receptor proteins, known as integrins, and plasminogen.

Cell‐cell contact activation of fibroblasts increases the expression of matrix metalloproteinases

Background. We recently found that direct homotypic cell‐cell contacts between human dermal fibroblasts induce a novel form of cell activation leading to non‐apoptotic programmed cell death. As the major features of this process we identified massive induction of cyclo‐oxygenase‐2 and production of inflammatory prostaglandins. On the surface of the decomposing spheroids, activation of the major extracellular proteolytic cascade, plasminogen activation, associated with surface exposure of α‐enolase, took place. Aim. To further characterize pericellular proteolysis by cell‐cell contact‐activated fibroblasts we studied the role of the other major extracellular proteolytic system, matrix metalloproteinases (MMPs). Methods. MMP expression in fibroblast clusters and monolayers was compared using mRNA microarrays and immunoblot analyses. The activities of MMPs were confirmed using MMP inhibitors and caseinolysis. Results. In microarrays MMP‐1, ‐10, and ‐14 (MT1‐MMP) were induced 5.8‐, 106‐, and 5.6‐fold, respectively. These findings were confirmed by immunoblotting. Radial caseinolysis showed low level of proteolytic activity in spheroid‐conditioned media; ilomastat, a general inhibitor of MMPs, suppressed 50% of the proteolytic activity thus confirming it to be at least in part due to MMPs. A cocktail of tetracycline‐derived MMP inhibitors suppressed lactate dehydrogenase (LDH) release only 11%, and if combined with aprotinin 28%. Conclusions. Cell‐cell contact activation of fibroblasts induced MMP‐1, ‐10, and MT1‐MMP expression, suggesting similar signaling to that in inflammation and cancer.

Active transforming growth factor-β in human melanoma cell lines: No evidence for plasmin-related activation of latent TGF-β

Cultured human melanoma cells were found to secrete TGF‐β mostly in latent biologically inactive form but in addition five of six melanoma cell lines studied produced in conditioned culture medium active TGF‐β in the range from 370 to 610 pg per 106 cells per 24 h. A distinct characteristic of these melanoma cell lines is that they form active surface‐bound plasmin by the activation of plasminogen with surface‐bound tissue‐type plasminogen activator. The present study was performed to assess the role of plasmin in the process of latent TGF‐β activation in the melanoma cell lines. No direct correlation was found between cell‐associated plasmin activity and the amount of active TGF‐β present in the conditioned medium of individual cell lines. The melanoma cell lines exhibited diverse responses to exogenous active TGF‐β1; three cell lines were growth‐stimulated, two were growth‐inhibited, and one had a very low sensitivity to the growth factor. The active TGF‐β produced by the melanoma cells was found to inhibit the natural killer cell function of peripheral blood lymphocytes, suggesting that it may have an immunosuppressive effect and a role in the development of melanomas.