Resumo

Laminin (LM), an extracellular matrix glycoprotein, plays a pivotal role in central nervous system (CNS) development and regeneration. The laminin molecule, which is arranged in polymers in vivo and is able to self-polymerize in vitro, regulates several cellular functions in CNS, such as survival, proliferation, migration and differentiation. We have previously shown that laminin matrices polymerized in acidic buffer (LM4) display a homogeneous structural organization, which is not observed in matrices polymerized in neutral buffer (LM7). We have also shown that neural cells can recognize and distinguish between the two types of LM polymers. The aim of this work is to investigate the intracellular pathways that mediate the response to LM polymers. In particular we will explore the possibility that the geometry of the polymers contributes to their signaling properties. A single cell suspension of rat neural cells, derived from cortex (E14) or retinal tissue (P1), was seeded on artificial LM matrices polymerized at pH 4.0 (LM4), at pH 7.0 (LM7) or on poly-L-ornithine (PLO). After 24 hours, the cells were fixed, immunolabeled and analyzed using phase-contrast or confocal fluorescence microscopy. In order to investigate signal transduction, inhibitors of the kinases MAPK (PD98059), PKA (H89) and PKC (queleritrin chloride) were used, as well as Rolipram and Forskolin, which increase cAMP levels. These inhibitors were also used in cell proliferation and differentiation assays. In order to explore the contribution of the polymer geometry, we used mimetic matrices of polyethersulfone simulating the surfaces of LM4 or LM7 covered with non-polymerized laminin. When platted on LM4 cells are arranged in monolayers where neurons emit long processes evenly distributed over the substrate. On the other hand, cultures established on LM7 showed the formation of cell clusters, where cellular extensions are apparently confined to the clusters. Similar results are obtained for cells platted on the respective mimetic matrices. Quantitative analysis reveals an increase of ~100% in the incorporation of thymidine when the cells are plated on LM4 when compared to LM7. Treatment with H89, which blocks activation of PKA, decreases cell proliferation only on LM4. In contrast, inhibitors of PKC and MAPK do not affect proliferation on LM4. In addition, we show that inhibition of PKA suppressed neurite outgrowth in LM4, while outgrowth is increased by Forskolin or Rolipram. Similar results are obtained for either retinal or embryonic cortical cells. Our data suggest that LM4 specifically stimulate the cAMP/PKA pathway on neural cells and that the signaling properties of the laminin matrices are at least partially due to their topology.