Cell-to-cell and cell-to-extracellular matrix (ECM) adhesions are essential for the development and function of multicellular organisms. The neural cell adhesion molecule (NCAM), abundantly expressed in the nervous system and in tumour cells, modulates events such as migration, differentiation and synaptic plasticity (Bonfanti 2006) by operating as a signalling receptor-like structure for homophilic and heterophilic inputs (Hinsby et al. 2004). NCAM is subject to an unusual post-translational modification, polysialic acid (PSA). PSA plays a modulatory role in NCAM-mediated adhesions by promoting NCAM binding with ECM-components (Hinsby et al. 2004). In contrast, non-polysialylated-NCAMs display predilection for homophilic -cis-trans NCAM-NCAM adhesions (Soroka et al. 2003). Accordingly, PSA acts as a switch between cell-to-cell and cell-to-ECM interactions and in doing so adjusts NCAM function from adhesion to signalling (Gascon et al. 2007; Senkov et al. 2012).
Despite understanding the importance of PSA–NCAM, regulation of cell surface PSA–NCAM remains a poorly understood process with modulation of the expression and/or activity of the two Golgi-associated polysialyltransferases, ST8siaIV (PST) and ST8siaII (STX) (Eckhardt et al. 1995) considered the main regulatory mechanism. The developmental regulation of PST and STX is correlated with that of PSA–NCAM at the cell surface (Bonfanti 2006). The activity of PST/STX is strongly dependent on intracellular stores of Ca2+ and, thus, regulated by perturbations in the concentrations of this ion (Bruses and Rutishauser 1998). Consequently, developmental up-regulation of NMDAR, and subsequent activation of Ca2+ signalling pathways, is associated with a reduction in PSA levels in adult brainstem synapses (Bouzioukh et al. 2001a). Based on the premise that PSA at the cell surface is the resulting net balance between PSA–NCAM synthesis and turnover we demonstrate that PST/STX enzymatic activity per se does not modulate cell-surface PSA–NCAM. Instead, our results demonstrate that PSA–NCAM synthesis and turnover are controlled by different mechanisms which, being independently regulated, allow for fine-tuning of cell-surface PSA–NCAM.
The ECM constitutes a 3D physical-scaffold which plays critical roles in tissue morphogenesis and homeostasis (Frantz et al. 2010). Constant cell–ECM interactions account for the heterogeneous and highly dynamic remodelling of the extracellular milieu and for modulation of key cellular events. The dynamic role of the ECM on the biology of the cells is particularly illustrated by the interaction of integrin-family members with specific components of the extracellular milieu. This in turn activates endocytic/exocytic receptor trafficking and intracellular signalling pathways aimed at remodelling the extracellular microenvironment and induce cytoskeleton rearrangements required for cell migration (Caswell et al. 2009; Huttenlocher and Horwitz 2011). Cell–ECM interactions are influenced by a number of mechanisms, including growth factor signalling (Rozario and DeSimone 2010), and implicated, among other cellular events, in differentiation and migration in neurons, invasion and metastasis in tumour (Guvakova 2007; Sachdev and Yee 2007).
In this study, turnover of PSA–NCAM was investigated in human rhabdomyosarcoma TE671 cells (Daniel et al. 2001). We provide evidence that PSA–NCAM turnover is principally achieved by endocytosis, induced upon interactions with ECM-components such as collagen type IV and modulated by insulin-like growth factor 1 (IGF1) and insulin. Induction of PSA–NCAM turnover promoted accumulation of non-polysialylated-NCAMs and, thereby, a switch in the NCAM-mediated adhesion mode. The results of this study reveal a novel role for IGF1 and insulin in modulation of cell-surface PSA–NCAM which has functional implications for PSA in cell migration (Rutishauser 2008; Brennaman and Maness 2010) and tumour cell metastasis (Amoureux et al. 2010).
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The balance between PSA–NCAM and NCAM at the cell surface is critical for cell migration and synaptic plasticity (Rutishauser 2008). Cell surface PSA–NCAM was proposed to be a direct function of its synthesis rate (Soares et al. 2000; Angata and Fukuda 2003); however, in TE671 cells, inhibition of PSA–NCAM synthesis is a necessary but not sufficient condition to reduce cell-surface PSA–NCAM. Turnover of PSA–NCAM was achieved when its synthesis was inhibited (Bruses and Rutishauser 1998) but only upon activation of PSA–NCAM internalization by the ECM. Thus, cell-surface PSA–NCAM/NCAM ratios in TE671 cells are the resulting net balance between two major mechanisms, synthesis and turnover, independently regulated but acting in tight coordination (Bonfanti 2006).
Cell lysates cultured in ECM did not show changes in PSA–NCAM or was PSA detected in culture media or ECM samples, thus posing PSA–NCAM desialylation as an intracellular mechanism involving internalization of the molecule in TE671 cells. Furthermore, the effective prevention of PSA–NCAM turnover by endocytosis inhibitors and the co-localization of PSA–NCAM with Rab5 corroborated the involvement of the endocytic pathway in PSA–NCAM turnover (Bouzioukh et al. 2001b; Minana et al. 2001). Shed NCAM-ectodomains were previously reported devoid of PSA (Hinkle et al. 2006; Brennaman and Maness 2008) and our data concurs that MMP-mediated proteolysis of NCAM occurs after desialylation of PSA–NCAM.
Recycling of adhesion receptors such as integrins via the endosomal pathway promotes the dynamics required for cell migration (Bretscher 2008; Huttenlocher and Horwitz 2011). In concurrence with previous studies (Bouzioukh et al. 2001a; Minana et al. 2001; Diestel et al. 2007), our data pose modulation of PSA–NCAM/NCAM at the cell surface as a recycling process, whereby NCAMs are polysialylated at the Golgi and transported to the cell surface. Subsequently, cell surface PSA–NCAMs are internalized into acidic endosomes where PSA is degraded, because of the labile glycosidic linkages of sialic acids in low pH environments (Manzi et al. 1994), and NCAMs sorted for re-polysialylation at the Golgi and transported to the cell surface. In this scenario, inhibition of NCAM-polysialylation results in de novo synthesized and/or sorted non-polysialylated-NCAMs being transported to the cell surface, where they are now susceptible to MMP-mediated ectodomain shedding (Hinkle et al. 2006), while cell-surface PSA–NCAM is continuously being internalized into the cytosol. The continuous supply of non-polysialylated-NCAMs, in combination with the continuous internalization of PSA–NCAM, leads to increased non-sialylated-NCAM at the cell surface and consequently to a progressive switch in the NCAM-mediated interaction status of the cell.
PSA exerts steric impediment between cells and modulates NCAM-mediated interactions (Rutishauser 2008). Our results showed a PSA-dependent pivotal shift in the MW of NCAM suggesting the establishment of NCAM-interactions with ligands other than PSA and, thereby, a switch in the overall NCAM-interaction mode. In the absence of PSA, NCAM displays stronger affinity for homophilic interactions (Hinsby et al. 2004), thus our results might reflect the formation of large zipper-like NCAM-NCAM complexes in cis/trans (Soroka et al. 2003). Alternatively, a number of partners have been described for heterophilic NCAM-interactions such as L1, FGF and glutamate receptors, HSPG and collagens (Storms and Rutishauser 1998; Gascon et al. 2007; Senkov et al. 2012). This poses the possibility for non-polysialylated-NCAMs in our cellular model interacting with ECM- or cellular components, the latter requiring ECM-components such as HSPGs for modulation of their strength (Nielsen et al. 2010), and forming large structural complexes.
Dysregulated NCAM-mediated adhesions are remarkably associated with non-polysialylated-NCAMs and correlate with gross brain-wiring abnormalities in mouse models (Brennaman and Maness 2008; Hildebrandt et al. 2009). Accordingly, our results showing a significant down-regulation for NCAM gene expression (Figure S4) suggest the activation of a negative-feedback regulatory mechanism for NCAM in conditions inducing the production of non-polysialylated-NCAMs [i.e. polysialylation of NCAM is inhibited: CI-medium or up-regulation of NMDAR in vivo (Bouzioukh et al. 2001b)]. Such mechanisms would avoid excessive accumulation of non-polysialylated-NCAM at the cell surface preventing unregulated NCAM-mediated interactions with potentially severe consequences (Weinhold et al., 2005).
Our results point toward ECM-components acting as a trigger-switch in a ligand-receptor endocytosis-mediated PSA–NCAM turnover. Using ECMs of different composition, we identified a novel function for collagen type IV and IGF1/insulin as respective inducer and inhibitors of PSA–NCAM internalization in TE671 cells. IGFs in the ECM play functional roles in events such as turnover of cell-adhesions (Guvakova 2007). IGF1R forms dynamic complexes with αv-integrin which are disrupted upon IGF1 interaction causing integrin relocalization and a subsequent shift from cell-to-cell to focal contacts that promotes cell migration (Canonici et al. 2008). We did not detect such structural interaction between PSA–NCAM and IGF1R (data not shown); however, the formation of PSA–NCAM–IGF1R complexes may be weak or transient, or mediated through an unknown linker protein in TE671 cells. Further research is required to unravel the functional cross-talk between IGF1R and the turnover of PSA–NCAM; however, we speculate that likely pivotal-candidates for IGF1R-PSA–NCAM cross-talk are integrin-family members. NCAM is known to interact with β1-integrin (Petridis et al. 2011), involved in modulation of integrin-dependent cell migration (Diestel et al. 2005) and, similar to L1, proposed to act as co-endocytosis carrier for integrin-family members and in recycling of focal adhesions and integrin-dependent migration processes (Diestel et al. 2007; Schmid and Maness 2008). Interaction between components of the ECM, such as collagen, and integrin-family members leads to endosomal trafficking of several molecules and to the establishment of polarized/asymmetric adhesions promoting cell migration (Hynes 2002; Caswell et al. 2009; Huttenlocher and Horwitz 2011). The implication of collagen type IV in PSA–NCAM turnover in TE671 cells also suggests the involvement of integrin-family members in the PSA–NCAM internalization process.
Modulation of cellular responses by the ECM is largely established as a direct function of its dynamic topology and biochemical composition (Frantz et al. 2010). Moreover, our results suggest a functional association between the in vivo dynamics of the ECM biochemical composition and the balance of PSA–NCAM/NCAM affecting key cellular events such as development and migration (Rutishauser 2008). Increasing evidence from clinical and pre-clinical studies pose IGF1/insulin as key players in CNS neuroplasticity (Reagan 2010). Our data suggest an interesting functional correlation between IGF1/insulin, PSA–NCAM turnover, synaptic dysfunctions and neurodegenerative diseases associated with dysregulated PSA–NCAM interactions such as Alzheimer's and schizophrenia (Hildebrandt et al. 2009; Brennaman and Maness 2010).