Determinants of differential functions of galectin-8 on T cells and tumoral cells : interplay of galectin-8 isoforms, integrin and growth factor signaling pathways.
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2014
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Abstract
Galectins are a subfamily of lectins involved in a variety of cellular processes mainly related with the immune system and cancer. Galectins are secreted through unconventional mechanisms, because they lack a signal peptide, and their carbohydrate recognition domains (CRD) bind to N-acetyllactosamine residues present on cell surface N- and O-glycoconjugates. Each of the 15 galectin family members can have redundant as well as particular functions depending on the structural organization of their CRDs, which is classified in three major groups: Monomeric chimera-type design (Gal-3); Homodimeric prototype (Gal-1, -2, -5, -7, -10, -11, -13, and -15); Tandem-repeat type with two different CRDs connected by a linker peptide (Gal-4, -6, -8, -9 and -12). The function of each particular member can also vary in different cellular contexts through mechanisms still not well understood, but likely dependent on differential subsets of glycoproteins and signaling pathways engaged. Therefore, understanding the role of a particular galectin requires to define the determinants of its variable functions in different cellular contexts.
This thesis studies the function of Gal-8, one of the most widely expressed galectins, both in normal tissues and cancerous cells. Gal-8 has been shown to induce apoptosis in activated T cells from freshly isolated peripheral blood mononuclear cells (PBMC), an effect suggesting a suppressive role in the immune system. In addition, unpublished data from our laboratory shows that Gal-8 increases proliferation, migration and invasion of normal epithelial MDCK cells, activating the Epidermal Growth Factor Receptor (EGFR) (PhD thesis of Claudia Oyanadel). Such an effect might be important in the pathogenesis of tumoral cells, as many kinds of cancers overexpress this lectin. The determinants of such a distinct Gal-8 effects are unknown but likely depends o differential engagement of signaling pathways. An additional level of control of Gal-8 function might depend on particular isoforms. As a tandem-repeat type, Gal-8 has two distinct CRD linked by a peptide that varies in size giving rise to different isoforms. The linker of Gal-8 medium/standard isoform (Gal-8S) has 34 amino acids while Gal-8 long isoform (Gal-8L) has 42 additional residues that include a thrombin cleavage site. Most of the studies, including the effects already mentioned, have focused on Gal- 8S and it is currently unknown whether Gal-8L has a redundant or distinct function.
Here we asked two main related questions: 1) whether the proliferation pathway induced by Gal-8 always depend on EGFR activation, or can involve another signaling pathway, and; 2) whether the linker size of Gal-8 isoforms has functional implications. To answer these questions we initially compared the effects of Gal-8S and Gal-8L on the pathways leading to apoptosis in Jurkat T cells and to proliferation in tumoral HeLa cells overexpressing the EGFR. The results lead to include studies on the effects of separate N- and C-terminal CRDs, which can be proteolitically released from Gal-8L, on apoptosis, migration and proliferation. The hypothesis is: \201CGal-8 function varies accordingly to its operating isoform and the potential to engage different signaling pathways depending on the cell context\201D. We show that: (i) Gal-8S apoptotic effect of Jurkat T cells depends on the activation of of \03B21-integrin pathway leading to ERK1/2 activation. Gal-8L, as well as separate carbohydrate recognition domains (CRDs), counteracts the pro-apoptotic activity of Gal-8S in Jurkat T cells.
Gal-8L diminished the Gal-8S-induced ERK1/2 activation and activated the anti-apoptotic AKT pathway. Although both isoforms interacted with and induced endocytosis of \03B21-integrin only Gal-8S induced its activation. Gal-8L and CRD pre-treatments also inhibited \03B21-integrin activation induced by Gal-8S. (ii) Gal-8 isoforms have differential effects on the proliferation and similar effects on migration of HeLa and U87 cells. While Gal-8S induced cell proliferation in both cell types, Gal- 8L and CRDs diminished the proliferation in HeLa cells and had no effect on U87 cells. Both Gal-8S and Gal-8L either inhibits or increase HeLa and U87 cell migration depending on the lectin presentation. Pre-binding of Gal-8 to cells in suspension inhibits cell migration while adding Gal-8 after cell attachment to substrate increases migration. (iii) Gal-8S binds and activates the EGFR and induces its endocytosis. Although it is very suggestive we do not known whether these effects are causally related.
(iv) Gal-8S induced HeLa cell proliferation involves activation of Sonic Hedgehog but not EGFR activation, even though both pathways become activated. Activation of Shh was demonstrated by Gal-8S induced expression of Shh target genes such as Gli-1 and Patched. These effects contrast with the previous observations on MDCK cells proliferation that does require EGFR activation. In conclusion, both length-linker isoforms and differential engagement of signaling pathways determines distinct cell responses to Gal-8.
This thesis studies the function of Gal-8, one of the most widely expressed galectins, both in normal tissues and cancerous cells. Gal-8 has been shown to induce apoptosis in activated T cells from freshly isolated peripheral blood mononuclear cells (PBMC), an effect suggesting a suppressive role in the immune system. In addition, unpublished data from our laboratory shows that Gal-8 increases proliferation, migration and invasion of normal epithelial MDCK cells, activating the Epidermal Growth Factor Receptor (EGFR) (PhD thesis of Claudia Oyanadel). Such an effect might be important in the pathogenesis of tumoral cells, as many kinds of cancers overexpress this lectin. The determinants of such a distinct Gal-8 effects are unknown but likely depends o differential engagement of signaling pathways. An additional level of control of Gal-8 function might depend on particular isoforms. As a tandem-repeat type, Gal-8 has two distinct CRD linked by a peptide that varies in size giving rise to different isoforms. The linker of Gal-8 medium/standard isoform (Gal-8S) has 34 amino acids while Gal-8 long isoform (Gal-8L) has 42 additional residues that include a thrombin cleavage site. Most of the studies, including the effects already mentioned, have focused on Gal- 8S and it is currently unknown whether Gal-8L has a redundant or distinct function.
Here we asked two main related questions: 1) whether the proliferation pathway induced by Gal-8 always depend on EGFR activation, or can involve another signaling pathway, and; 2) whether the linker size of Gal-8 isoforms has functional implications. To answer these questions we initially compared the effects of Gal-8S and Gal-8L on the pathways leading to apoptosis in Jurkat T cells and to proliferation in tumoral HeLa cells overexpressing the EGFR. The results lead to include studies on the effects of separate N- and C-terminal CRDs, which can be proteolitically released from Gal-8L, on apoptosis, migration and proliferation. The hypothesis is: \201CGal-8 function varies accordingly to its operating isoform and the potential to engage different signaling pathways depending on the cell context\201D. We show that: (i) Gal-8S apoptotic effect of Jurkat T cells depends on the activation of of \03B21-integrin pathway leading to ERK1/2 activation. Gal-8L, as well as separate carbohydrate recognition domains (CRDs), counteracts the pro-apoptotic activity of Gal-8S in Jurkat T cells.
Gal-8L diminished the Gal-8S-induced ERK1/2 activation and activated the anti-apoptotic AKT pathway. Although both isoforms interacted with and induced endocytosis of \03B21-integrin only Gal-8S induced its activation. Gal-8L and CRD pre-treatments also inhibited \03B21-integrin activation induced by Gal-8S. (ii) Gal-8 isoforms have differential effects on the proliferation and similar effects on migration of HeLa and U87 cells. While Gal-8S induced cell proliferation in both cell types, Gal- 8L and CRDs diminished the proliferation in HeLa cells and had no effect on U87 cells. Both Gal-8S and Gal-8L either inhibits or increase HeLa and U87 cell migration depending on the lectin presentation. Pre-binding of Gal-8 to cells in suspension inhibits cell migration while adding Gal-8 after cell attachment to substrate increases migration. (iii) Gal-8S binds and activates the EGFR and induces its endocytosis. Although it is very suggestive we do not known whether these effects are causally related.
(iv) Gal-8S induced HeLa cell proliferation involves activation of Sonic Hedgehog but not EGFR activation, even though both pathways become activated. Activation of Shh was demonstrated by Gal-8S induced expression of Shh target genes such as Gli-1 and Patched. These effects contrast with the previous observations on MDCK cells proliferation that does require EGFR activation. In conclusion, both length-linker isoforms and differential engagement of signaling pathways determines distinct cell responses to Gal-8.
Description
Tesis (Doctor en Ciencias Biológicas, mención en Biología Celular y Molecular)--Pontificia Universidad Católica de Chile, 2014