In search for a cheap and effective transfection reagent we used the positively charged polyplexforming compound polyethylenimine (PEI). This compound is commercially available from different companies either as a non-modified chemical reagent or with additives as a more cost intensive transfection reagent. Here we used the non-modified PEI reagent to optimize transfection protocols for different cell-lines. With these optimized conditions we were able to transiently transfect a number of cell-lines up to 40 -90%.

After skin wounding, the repair process is initiated by the release of growth factors, cytokines, and bioactive lipids from injured vessels and coagulated platelets. These signal molecules induce synthesis and deposition of a provisional extracellular matrix, as well as fibroblast invasion into and contraction of the wounded area. We previously showed that sphingosine-1-phosphate (S1P) triggers a signal transduction cascade mediating nuclear translocation of the LIM-only protein Fhl2 in response to activation of the RhoA GTPase (Muller, J.M., U. Isele, E. Metzger, A. Rempel, M. Moser, A. Pscherer, T. Breyer, C. Holubarsch, R. Buettner, and R. Schule. 2000. EMBO J. 19:359–369; Muller, J.M., E. Metzger, H. Greschik, A.K. Bosserhoff, L. Mercep, R. Buettner, and R. Schule. 2002. EMBO J. 21:736–748.). We demonstrate impaired cutaneous wound healing in Fhl2-deficient mice rescued by transgenic expression of Fhl2. Furthermore, collagen contraction and cell migration are severely impaired in Fhl2-deficient cells. Consequently, we show that the expression of α-smooth muscle actin, which is regulated by Fhl2, is reduced and delayed in wounds of Fhl2-deficient mice and that the expression of p130Cas, which is essential for cell migration, is reduced in Fhl2-deficient cells. In summary, our data demonstrate a function of Fhl2 as a lipid-triggered signaling molecule in mesenchymal cells regulating their migration and contraction during cutaneous wound healing.

We have described the scaffolding protein FHL2 as a component of focal adhesion structures, to which it is recruited via binding to both alpha- or beta-integrin subunits. Using mesenchymal stem cells from wild-type and FHL2-knockout mice, we show here that inactivation of FHL2 leads to impaired assembly of extracellular matrix proteins on the cell surface and to impaired bundling of focal adhesions. Both altered properties can be restored by reexpression of recombinant FHL2 protein in FHL2-null cells. Molecular analysis of integrin-mediated signaling revealed a higher phosphorylation of FAK at tyrosine 925 in FHL2-knockout cells compared to their wild-type counterpart. Consequently, the activation of the mitogenic kinase ERK was more pronounced in knockout cells on cell adhesion. The growth factor-induced activation of ERK, however, was not altered. The perturbed organization of extracellular matrix on FHL2-null cells was improved when the increased activation of MAPK was inhibited. Our findings point to a role of FHL2 in bundling of focal adhesion structures, in integrin-mediated ERK activation, and subsequently in proper allocation of matrix proteins on the cell surface.

Integrin ␣7␤1 is the major laminin binding integrin receptor of muscle cells. The ␣7 chain occurs in several splice isoforms, of which ␣7A and ␣7B differ in their intracellular domains only. The fact that the expression of ␣7A and ␣7B is tightly regulated during skeletal muscle development suggests different and distinct roles for both isoforms. However, so far, functional properties and interacting proteins were described for the ␣7B chain only. Using a yeast two-hybrid screen, we have found that Def-6, a guanine nucleotide exchange factor for Rac1, binds to the intracellular domain of the ␣7A subunit. The specificity of the Def-6-␣7A interaction has been shown by direct yeast two-hybrid binding assays and coprecipitation experiments. This is the first description of an ␣7A-specific and -exclusive interaction, because Def-6 did not bind to any other tested integrin cytoplasmic domain. Interestingly, the binding of Def-6 to ␣7A was abolished, when cells were cotransfected with an Src-related kinase, which is known to phosphorylate Def-6 and stimulate its exchange activity. We found expression of Def-6 was not only restricted to T-lymphocytes as described thus far but in a more widespread manner, including different muscle tissues. In cells, Def-6 is seen in newly forming cell protrusions and focal adhesions, and its localization partially overlaps with the ␣7A integrin receptor. C2C12 myoblasts overexpressing Def-6 show a delay of Rac1 inactivation during myogenic differentiation and abnormal myotube formation. Thus, our data suggest a role for Def-6 in the fine regulation of Rac1 during myogenesis with the integrin ␣7A chain guiding this regulation in a spatio-temporal manner.

In four independent yeast two-hybrid screens with the integrin alpha-subunits alpha3A, alpha6A, alpha7A, and alpha7B, we identified the Mss4 protein, a nucleotide exchange factor for exocytic Rab GTPases, as a novel integrin interacting protein. We have previously shown that it binds to the conserved KXGFFKR region of integrin alpha-subunits located directly beneath the cell membrane. Here we show that the binding site for integrins on Mss4 is overlapping with those for Rab GTPases. Functional analysis of the Mss4/integrin interaction revealed its importance for activation of matrix metalloproteinases (MMPs) and remodeling of secreted extracellular matrix (ECM) proteins. The exocytosis of all the proteins analyzed, however, was unaffected. Furthermore, our data suggest that Mss4 drives the coordinated action of the MT1-MMP/integrin protein complex, thus regulating the presence and activation of MT1-MMP at newly formed filopodia and lamellipodia. This in turn facilitates the conversion of pro-MMPs to MMPs, resulting in cleavage and remodeling of ECM proteins. C2C12 myoblasts with stably down-regulated Mss4 showed a disturbed fibronectin remodeling during differentiation, resulting in malfunctioned myotube formation.