During T cell development, bone marrow-derived precursors migrate towards the thymus. Developing thymocytes travel within the cortex and medulla through cell-cell and cell-extracellular matrix contacts and also in response to soluble secretory moieties such as cytokines and chemokines [1, 2]. During this process, there is a requirement of several time-dependent adhesion events between thymocytes and cells of the thymic microenvironment. Among the various molecular mechanisms involved in such heterocellular adhesion, one is represented by fibronectin/integrin mediated interactions. In particular, previous studies using anti-CD49e blocked antibody have shown that such an adhesion is partially under control of the FN/VLA-5 ligand/receptor pair [4, 13–15].
Herein, we applied the RNA interference strategy to silencing the CD49e gene, thus disrupting the integrin VLA-5 in human thymic epithelial cells. Although being transient, in most experiments we got a high efficiency in abrogating the corresponding CD49e mRNA, as well as its translation into the α5 integrin subunit (as ascertained by its detection on the cell membrane). Moreover, the specificity of the siRNA knockdown of the CD49e gene was ascertained by the fact that no effect was seen with a scramble siRNA. Also, it is interesting that the expression of CD49d (α4 integrin subunit of the VLA-4 fibronectin receptor) quantified by a specific antibody for the CD49d molecule using flow cytometry, remained unchanged in CD49e-siRNA-treated TEC. One could argue that another CD49d-containing integrin, namely α4β7 integrin might be modulated by CD49e knock down. However, this is not the case, since the VLA-4 integrin (CD49d/CD29) is actually the only CD49d-containing integrin constitutively expressed by this human thymic epithelial cell preparation, as previously ascertained by immunoprecipitation and cytofluorometry . In a second vein, although the expression of CD29 mRNA (the β1 subunit that forms the various members VLA integrin sub-family) also remain unchanged, we cannot discard the possibility that other members of the VLA family (for example the laminin receptor are modulated in this experimental conditions. Yet, in any case, from a conceptual point of view our findings unravel the independency of the regulation of genes coding for different ECM receptors.
It should be point out, however, other molecules related to cell adhesion/migration can be somewhat under control of CD49e. In this respect, we found a down-regulation of the gene coding for another adhesion receptor, namely neuronal cell adhesion molecule (NrCAM), which belongs to the L1 family of immunoglobulin-like cell adhesion molecules, initially discovered in nervous system . In this regard, it is been described that NrCAM interacts with various integrins including α5β1, and that such interactions can support cell binding and migration .
NrCAM is part of a growing list of “neural” molecules, also including neuropilins, which are expressed in immune systems, including the thymus [see reviews] [19, 20]. In addition to neuropilins, plexin A (that mediates neuropilin-induced intracellular signaling), and VEGF, one of neuropilin natural ligands have been described in the thymus . In the human thymus, neuropilins mediate thymocyte migration [19, 20]. All these genes are down-regulated following siRNA-induced CD49e knockdown in human TEC, strongly indicating the existence of a highly complex intracellular molecular circuitry in the thymic epithelium, controlling the expression of various genes involved in physiological guidance of developing thymocytes within the thymic lobules. It is conceivable that the mechanisms underlining such control may comprise direct as well as indirect effects of CD49e gene knockdown, as for example via modulation of specific microRNAs that target those genes, down- or up-regulated after CD49e silencing. Dissecting this circuitry shall provide a much better understanding of how the TEC network controls thymocyte migration and adhesion. In a second vein, the microarray analysis revealed that siRNA-induced knockdown of CD49e in cultured human TEC also deregulated the expression of signal transduction genes, some of them related with intracellular pathways triggered by integrins. The binding of fibronectin to α5β1 activates two major tyrosine kinase-dependent pathways, the focal adhesion kinase (FAK) pathway and the Shc pathway . FAK is recruited to the ligand binding site and becomes activated through phosphorilation of tyrosine residues . In turn, activation of FAK directly phosphorylates Src-family protein-tyrosine kinases . Additionally, FAK can form an association with phosphatidylinositol 3-kinase (PI3K), and such an association activates PI3K and its signaling pathways , regulating, among other functions, the actin cytoskeleton . The regulation of actin fibers by PI3K can be mediated by centaurin α-1, a phosphatidylinositol interacting protein, which directly interacts with F-actin, resulting in a decrease in stress fibers, as ascertained in Hela cells .
In our microarray data, we noticed that siRNA-induced CD49e gene silencing in human TEC resulted in up-regulation of the centaurin α-1 gene. Moreover, the down-regulation of phosphatidylinositol-4-phosphate 5-kinase type I (PIP5K) mRNA, that is the predominant kinase that synthesized phosphatidylinositol 4,5-bisphosphate [27, 28] also indicates a decrease in stress fibers in TEC with CD49e knocked down. Although we have not directly tackled this issue, such a decrease in F-actin could explain, at least partially, the decrease in the ability of CD49e-siRNA-treated TEC to promote thymocyte adhesion.
Among genes down-regulated in human cultured TEC in which CD49e was knocked down by RNA interference, we also found the activin receptor-like kinase 1 gene (acvrl1), which encodes a type I cell-surface receptor (ALK1) for transforming growth factor-β1 (TGF-β1). This cytokine is involved in the control of T cell development through direct effect on both thymocyte and epithelial cell compartments . Thus, it is conceivable that integrity of VLA-5 in human TEC is necessary to maintain proper levels one TGF-β receptor, thus allowing cross-talk between developing thymocytes and TEC.
Our results point some genes differential express due the CD49e silencing, however we can’t discard the effect of integrin crosstalk, a mechanism in which one integrin regulates the activation state of a different integrin in the same cell and this phenomenon involve the cytoskeleton associated protein talin . In addition, the integrin crosstalk may be at the level of regulation the stability of integrin subunit mRNA . We are just beginning to understand the molecular mechanisms that are involved in regulating the activation state of integrins and the molecular basis of crosstalk.
Finally, our data clearly show that knocking down the CD49e in human TEC, using RNA interference, results in a significant decrease in the ability of these cells to allow thymocyte adhesion. Such a decrease comprised all CD4/CD8-defined thymocyte subsets, a finding that is in accordance with the data showing that variable amounts of VLA-5 are expressed throughout the stages of intrathymic T cell differentiation . Whether other integrin-mediated TEC/thymocyte adhesion interactions are also modulated after CD49e gene knockdown (for example the VLA-6/laminin binding) is presently under investigation.