Integrin-mediated regulation of cell survival and cytokine/chemokine gene expression in human thymic epithelial cells cultured in vitro.

Starting date
March 1, 2002
Duration (months)
24
Managers or local contacts
Tridente Giuseppe
Keyword
INTEGRINS, CYTOKINES, SIGNALING, SURVIVAL, TRANSCRIPTION FACTORS, DIFFERENZIATIO

Integrins are a large family of transmembrane adhesion receptors able to recognize both extracellular matrix- or cellular ligands thereby activating multiple intracellular signaling pathways. As integrins lack enzymatic activity in the cytosolic portion their transduction potential depends on the association with adaptors and kinases proximal to the membrane which can in turn activate cytosolic families of kinases such as protein kinase C, small GTP-proteins of the Ras and Rho families, MAP and ERK kinases ( Defilippi et al, 1997,Giancotti and Ruoslahti 1999). Studies largely performed in adherent cell lines have underlined the master role exerted by integrins in the tissue homeostasis and cell-cell talking due to the finding that signals delivered and/or integrated by integrin and growth factor receptors regulate activation of transcription factors and expression of genes that can govern proliferation, survival, differentiation and production of cytokines or chemokines (Defilippi et al, 1997, Giancotti and Ruoslahti 1999, Mainiero et al 2000 ). We have previouly demonstrated in normal epithelial cells derived from thymus (TEC) that the recruitment of a3b1 or a6b4 integrins at the cell contact sites with thymocytes or their cross-linking with mAbs results in the activation of NF-kB and NF-IL6 transcription factors associated with gene expression of IL-6 ( Ramarli et al, 1998 ) or partial protection from apoptosis (Scupoli et al 2000). The ability of signals from integrins to activate the two transcription factors may be of particular interest in this cellular systems in the light of 1) their cooperation for the transactivation not only of IL-6 gene, but also of other genes coding for cytokines and chemokines involved in the thymocytes differentiation and 2) the regulatory activity exerted on the growth, the survival and the differentiation of complex and stratified epithelia (Beg and Baltimore, 1996, Scatena et al, 1998 , Diehl et al, 1998, Robinson et al, 1998, Oh et al, 1998).
The molecular mechanisms linking the intracellular kinase cascades triggered by integrins with the activation of NF-kB and NF-IL6 transcription factors still need to be elucidated in normal cells. However, it has been recently reported in tumor epithelial cell lines that MEKK or MAP kinases activated by cytokines or overexpressed by transfection can regulate the activation of NF-kB and NF-IL6 transcription factors as well as the processes of apoptosis and differentiation in which the activity of the two factors is known to be involved. In particular , it has been demonstrated that : a) MEKK1, MEKK2 and MEKK3 can phosphorylate IKKa/b thereby triggering the phosphorylation of IkBa/b that is required for nuclear translocation of NF-kB complexes (Zhao and Lee 1999, Lee at al 1998) b) p38 and ERK1/2 can increase the transactivation potential of NF-kB by phosphorylating its p65 subunit (Jefferies and O'Neill 2000, Carter et al 1999, Vanden Berghe et al 1998) c) p38 can phosphorylate the isoforms of NF-IL6 thus regulating the differentiation of tumor fibroblast cell lines or normal chondrocytes ( Engelman et al 1998, Zhen et al, 2000) d) p38 and ERK1/2 may modulate the apoptotic response , although the positive or negative effects may vary depending on the cell lineage, the stimulus or the farmacological inhibitor used to distinguish the selective involvement of one of the two kinases(Jefferies and O’Neill 2000, Lee et al 1998, Zhao and Lee 1999). As regard the NF-kB activation, additional mechanisms controlling its nuclear localization appear to include the phosphorylation of IkB alfa exerted by a new protein complex (signalosome) containing the JAB1 molecule , originally described as a co-activator of c-Jun and more recently as an interactor of the cytosolic portions of beta-1 and beta-2 integrins ( Bianchi et al , 2000, Seeger et al , 1998) . We have recently observed in the normal TEC that the cross-linking of a6b4 integrins induce the activation of Rac , MEKK3/6 and then of the three MAP kinases p38, JNK and ERK1 associated with IkB alfa degradation, NF-kB nuclear traslocation and NF-IL6 phosphorylation . Preliminary experiments of blockage with pharmacological inhibitors suggest that in these cellular system p38 exerts a dominant role with respect of ERK1 in the regulation of NF-kB traslocation, of the constitutive and inducible expression of IL-6, of the terminal differentiation “in vitro” and of the negative response to apoptotic stimuli. Based on these preliminary observations we propose to investigate in the TEC cellular system the activity of MEKK and MAP kinases activated by beta-1 and beta-4 integrins in the regulation of
A) the activation of NF-kB and NF-IL6 transcription factors and the expression of IL-6 and Rantes
B) the regulation of the caspase 9-dependent apoptotic response
C) the regulation of the terminal differentiation “in vitro”, also in cooperation with the activity of molecules that modulate the electric potential of the plasmamembrane ( Herg K+ channels)
The informations obtained will add to the kwnoledge on some of the molecular mechanisms regulated by integrins that contribute to the maintainance of the homeostasis of normal thymic epithelial tissuee and to the regulation of the production of cytokines and chemokines required for the proper maturation of T lymphocytes.

Sponsors:

Ministero dell'Istruzione dell'Università e della Ricerca
Funds: assigned and managed by the department
Syllabus: PRIN

Project participants

Valeria Antonini
Ornella Poffe
Technical-administrative staff

Collaboratori esterni

Monica Brentegani
Azienda Ospedaliera di Verona Tecnico di laboratorio

Activities

Research facilities