The molecular determinants underlying the failure of axons to regenerate in the CNS after injury were studied in an in vitro model of astrogliosis and neuronal coculture. Mechanically lesioned neuron-astrocyte mouse cortical cocultures were treated with antisense glial fibrillary acidic protein (GFAP)-mRNA in order to inhibit the formation of gliofilaments that occurs in response to injury. This inhibition relieves the blockage of neuron migration and neuritic outgrowth observed after lesion, and migrating neurons reappeared, supported by a laminin-labeled extracellular network (permissive conditions). We then questioned the relationship between this permissivity and laminin production. Follow-up studies on the concentration of laminin indicated that, after antisense treatment, the laminin level was increased in the cocultures and was under the control of astrocyte-neuron interactions. The addition of exogenous laminin favored neuronal migration and neurite outgrowth, whereas neutralizing laminin bioavailability with antibodies recognizing the astroglial laminin resulted in an inhibition of both neuronal access to the lesion site and neurite outgrowth, suggesting an active role for laminin in the permissive process. This permissive process could be associated with modulation of extracellular matrix (ECM) molecule degradation by proteinases. Among the latter, matrix metalloproteinases (MMPs) are involved in the breakdown of the ECM component. Our investigation showed a net decrease of the matrix metalloproteinase MMP-2 expression and activity and an increase of its endogenous inhibitor TIMP-2 expression. Both proteins associated with permissivity should be involved in the laminin stabilization and cell-matrix interactions. High levels of laminin and laminin bioavailability, consequent to a reduction in astrogliosis, may be important permissive elements for neuronal migration and neurite outgrowth postlesion.

The expression of glial fibrillary acidic protein (GFAP)-mRNA during mouse brain development and in astroglial primary cultures has been investigated by using two approaches: Northern-blot evaluation using a specific cDNA probe, and cell-free translation associated with immunoprecipitation. During brain maturation (4-56 days postnatal), the GFAP-mRNA underwent a biphasic evolution. An increase was observed between birth and day 15 (i.e., during the period of astroglial proliferation), which was followed by a decrease until day 56 (i.e., during astroglial cell differentiation). At older stages (300 days), an increase was observed, which might reflect gliosis. During astroglial in vitro development (7-32 days in culture), the GFAP-mRNA showed similar variations. An increase, observed during the period of astroglial proliferation (7-18 days), was followed by a decrease which occurred in parallel to marked changes in cell shape, cell process outgrowth, and the organization and accumulation of gliofilaments. During the same culture period (7-32 days), alpha-tubulin mRNA, which was used as an internal standard, did not vary significantly. These results show that the increase of the GFAP protein and of gliofilaments observed both in vivo and in vitro during astroglial differentiation cannot be ascribed to an accumulation of the GFAP-mRNA. It might be that more than one mechanism regulates the levels of free and polymerized GFAP and of its encoding mRNA.

(MMPs) present at the site of airway injury are thought to contribute to epithelial wound repair. As TGF-␤1 can modulate MMP expression and MMPs play an important role in wound repair, we hypothesized that TGF-␤1 may enhance airway epithelial repair via MMPs secreted by epithelial cells. We evaluated the in vitro influence of TGF-␤1 on wound repair in human airway epithelial cells cultured under conditions allowing differentiation. The results showed that TGF-␤1 accelerated in vitro airway wound repair, whereas MMP inhibitors prevented this acceleration. In parallel, we examined the effect of TGF-␤1 on the expression of MMP-2 and MMP-9. TGF-␤1 induced a dramatic increase of MMP-2 expression with an increased steadystate level of MMP-2 mRNA, contrasting with a slight increase in MMP-9 expression. To confirm the role of MMP-2, we subsequently evaluated the effect of MMP-2 on in vitro airway wound repair and demonstrated that the addition of MMP-2 reproduced the acceleration of wound repair induced by TGF-␤1. These results strongly suggest that TGF-␤1 increases in vitro airway wound repair via MMP-2 upregulation. It also raises the issue of a different in vivo biological role of MMP-2 and MMP-9 depending on the cytokine microenvironment.human nasal epithelial cells; matrix metalloproteinase-9; wound healing; cell migration HUMAN AIRWAY EPITHELIUM ACTS as a protective barrier and is subject to injury during exposure to a variety of inhaled toxins or infectious agents. Repair of epithelial injury requires a wound repair process involving multiple distinct steps, starting with spreading and migration followed by proliferation and differentiation of epithelial cells, which are accompanied by remodeling of the extracellular matrix (ECM) (7, 24).After injury, fibroblasts, inflammatory, endothelial, and epithelial cells produce various cytokines and growth factors including transforming growth factor-␤ (TGF-␤) (19,28). TGF-␤ exerts a wide spectrum of biological functions contributing to wound repair (13,15,19). In vitro, TGF-␤1 is known to modulate the composition of the provisional matrix over which the epithelial cells migrate (16) and to trigger behavioral changes in epithelial cells involved in wound repair (2). TGF-␤1 can also regulate the synthesis of certain matrix metalloproteinases (14,20,25). Matrix metalloproteinases (MMPs) form a family of zinc-dependent endopeptidases that are able to degrade the various macromolecular components of the ECM. Within the MMP family, MMP-2 (gelatinase A) and MMP-9 (gelatinase B) more specifically hydrolyze denatured collagens (gelatin) and native type IV collagen (29). MMP-2 and MMP-9 play a major role in physiological tissue remodeling processes, such as organogenesis and wound repair (10, 23). On the other hand, abnormal expression of these proteases is known to contribute to diverse disorders, including healing disorders, occurring in chronic inflammatory airway diseases (1,11,12). Although MMP-2 and MMP-9 share structural similarities, there is strong evidence that MMP-2 ...

The microenvironment of a tumor can influence both the morphology and the behavior of cancer cells which, in turn, can rapidly adapt to environmental changes. Increasing evidence points to the involvement of amoeboid cell migration and thus of cell blebbing in the metastatic process; however, the cues that promote amoeboid cell behavior in physiological and pathological conditions have not yet been clearly identified. Plasminogen Activator Inhibitor type-1 (PAI-1) is found in high amount in the microenvironment of aggressive tumors and is considered as an independent marker of bad prognosis. Here we show by immunoblotting, activity assay and immunofluorescence that, in SW620 human colorectal cancer cells, matrix-associated PAI-1 plays a role in the cell behavior needed for amoeboid migration by maintaining cell blebbing, localizing PDK1 and ROCK1 at the cell membrane and maintaining the RhoA/ROCK1/MLC-P pathway activation. The results obtained by modeling PAI-1 deposition around tumors indicate that matrix-bound PAI-1 is heterogeneously distributed at the tumor periphery and that, at certain spots, the elevated concentrations of matrix-bound PAI-1 needed for cancer cells to undergo the mesenchymal-amoeboid transition can be observed. Matrix-bound PAI-1, as a matricellular protein, could thus represent one of the physiopathological requirements to support metastatic formation.