A Protein Kinase C/Ras/ERK Signaling Pathway Activates Myeloid Fibronectin Receptors by Altering β1 Integrin Sialylation

Abstract

Here we report that myeloid cells differentiating along the monocyte/macrophage lineage down-regulate the ST6Gal-I sialyltransferase via a protein kinase C/Ras/ERK signaling cascade. In consequence, the ␤1 integrin subunit becomes hyposialylated, which stimulates the ligand binding activity of ␣5␤1 fibronectin receptors. Pharmacologic inhibitors of protein kinase C, Ras, and MEK, but not phosphoinositide 3-kinase, block ST6Gal-I down-regulation, integrin hyposialylation, and fibronectin binding. In contrast, constitutively active MEK stimulates these same events, indicating that ERK is both a necessary and sufficient activator of hyposialylationdependent integrin activation. Consistent with the enhanced activity of hyposialylated cell surface integrins, purified ␣5␤1 receptors bind fibronectin more strongly upon enzymatic desialylation, an effect completely reversed by resialylation of these integrins with recombinant ST6Gal-I. Finally, we have mapped the N-glycosylation sites on the ␤1 integrin to better understand the potential effects of differential sialylation on integrin structure/function. Notably, there are three N-glycosylated sites within the ␤1 I-like domain, a region that plays a crucial role in ligand binding. Our collective results suggest that variant sialylation, induced by a specific signaling cascade, mediates the sustained increase in cell adhesiveness associated with monocytic differentiation.The U937 and THP-1 cell lines represent well accepted model systems for studying myeloid differentiation along the monocyte/macrophage lineage. Following treatment with phorbol myristate acetate (PMA), 4 these cells exhibit phenotypic changes that are characteristic of cell differentiation, including increased respiratory burst activity, enhanced phagocytotic capability, and markedly elevated cell adhesiveness to extracellular matrix ligands such as fibronectin. In vivo, the increased adhesiveness of monocytes/macrophages contributes to the extravasation of cells from the vasculature as well as tethering of cells within inflamed tissues.Differentiating myeloid cells bind to fibronectin through the integrin family of cell adhesion receptors, including the ␣5␤1 integrin species. The molecular mechanisms underlying PMA-dependent cell adhesion have not been well defined, although it has been reported that PMA increases the synthesis of both ␣5 and ␤1 integrin subunits (1-4). However, myeloid cells (U937 and THP-1) express an abundant amount of ␣5␤1 in the absence of PMA treatment, and yet these cells bind very poorly to fibronectin. This suggests that myeloid ␣5␤1 receptors are normally in an inactive state and that increased expression alone cannot account for the dramatically increased fibronectin binding induced by PMA.In our prior study (5) we observed that PMA stimulated a rapid but transient increase in fibronectin binding that was likely due to the activation of integrins already present on the cell surface. However, following this initial transient event there was a second phase of elevated fibronect...

N-Glycosylation of the I-like Domain of β1 Integrin Is Essential for β1 Integrin Expression and Biological Function

“…2A, lower panel). It should be noted that the similar mobility on the SDS-PAGE between ⌬1-3 and ⌬7,8 mutants, or between ⌬4-6 and ⌬9-12 mutants, further supported the notion that site 2 and site 11 do not carry N-linked glycans as reported by Seales et al (33). The integrin ␤1 subunit associates with multiple ␣ subunits to form various heterodimers, which may stabilize its expression.…”

“…1. Reportedly, 10 of these sites are normally N-glycosylated, whereas sites 2 and 11 do not normally carry N-linked glycans (33). However, which of the 12 N-glycosylation sites are important for biological function of the subunit remains unknown.…”

Potential roles of N-glycosylation in cell adhesion

“…We found that N-glycosylation on the β-propeller domain of the α5 subunit (S3-5) is essential for its heterodimer formation and its biological functions such as cell spreading, cell migration and cytoskeletal formation, as well as for the proper folding of the α5 subunit [30]. Seales et al reported that the I-like domain on the β1 subunit, which could be the partner of the β-propeller of the α5 subunit [31], supporting the importance of N-glycans on the β-propeller. The crystal structure of integrin αVβ3 has been successfully revealed, and the main contact between the αV and β3 subunit is the β-propeller on the αV and A domain on β3 with hydrophobic, ionic, and other interactions [32,33].…”

Role for Hepatic and Circulatory ST6Gal-1 Sialyltransferase in Regulating Myelopoiesis

“…The humoral response (28 -30), maintenance of myeloid homeostatic balance (22), modulation of dendritic cell activity (31), T cell functionality (32), and integrin signaling (33)(34)(35) are but a few physiologic processes with implicated ST6Gal-1 participation. The idea of ST6Gal-1 pleiotropy Expression of (top left) total ST6Gal-1 mRNA, (top right) P1-transcribedST6Gal-1 mRNA that contains the unique Exon H sequence (2), and (bottom) selected hepatic sialyltransferases were measured by real-time PCR relative to the expression of RPL32, a ribosomal protein, as a reference standard.…”