Involvement of Galectin-3 with Vascular Cell Adhesion Molecule-1 in Growth Regulation of Mouse BALB/3T3 Cells

Abstract

␤-Galactose residues on N-glycans have been implicated to be involved in growth regulation of cells. In the present study we compared the galactosylation of cell surface N-glycans of mouse Balb/3T3 cells between 30 and 100% densities and found the ␤-1,4-galactosylation of N-glycans increases predominantly in a 100-kDa protein band on lectin blot analysis in combination with digestions by diplococcal ␤-galactosidase and N-glycanase. When cells at 100% density were treated with jack bean ␤-galactosidase, the incorporation of 5-bromodeoxyuridine into the cells was stimulated in a dose-dependent manner, suggesting the involvement of the galactose residues in growth regulation of cells. A galactose-binding protein was isolated from the plasma membranes of cells at 100% density by affinity chromatography using an asialo-transferrin-Sepharose column and found to be galectin-3 as revealed by mass spectrometric analysis. The addition of recombinant galectin-3 into cells at 50% density inhibited the incorporation of 5-bromodeoxyuridine in a dose-dependent manner, but the inhibition was prevented with haptenic sugar. An immunocytochemical study showed that galectin-3 is present at the surface of cells at 100% density but not at 30% density where it locates inside the cells. Several glycoproteins bind to a galectin-3-immobilized column, a major of which was identified as vascular cell adhesion molecule (VCAM)-1. Immunocytochemical studies showed that some galectin-3 and VCAM-1 co-localize at the surface of cells at 100% density, indicating that the binding of galectin-3 secreted from cells to VCAM-1 is one of the pathways involved in the growth regulation of Balb/3T3 cells.

Heterogenic Endothelial Responses to Inflammation: Role for Differential N ‐Glycosylation and Vascular Bed of Origin

“…Another possible candidate is VCAM‐1, as removal of sialic acid increases leukocyte trafficking, but this phenomenon was difficult to explain, as no N ‐glycan sites are found in proximity to the VLA‐4 binding domain . This gap has recently been clarified by studies demonstrating that galectin‐3 can bind to VCAM‐1 and support leukocyte adhesion . With PECAM‐1, α‐2,6‐sialic acid has been reported to be critical for homodimerization and associated antiapoptotic signaling .…”

“…It has been established that innate differences in surface carbohydrate patterns exists across vascular endothelium in vivo which is further modified during inflammation and disease . Moreover, it is now evident that endothelial cell expressed carbohydrates are involved in leukocyte trafficking, with these sugars providing a “zip code” to allow specific leukocyte subsets to migrate into specific tissues in response to specific stimuli . For example, our recent studies demonstrate a role for endothelial high mannose N ‐glycans in monocyte rolling/adhesion and suggest that N ‐glycosylation per se is regulated by inflammatory stimuli, by mechanisms that are distinct from those that affect adhesion molecule protein expression .…”

Vascular Cell Adhesion Molecule-1 Expression and Signaling During Disease: Regulation by Reactive Oxygen Species and Antioxidants

“…3). It has been reported that recombinant galectin 3 columns bind several proteins from BALB=3T3 cells, and the major band, at 100 kD, was identified as VCAM-1 by mass spectrometry (278). Moreover, VCAM-1 is immunoprecipitated from proteins bound to a recombinant galectin-3 column (278).…”

Galectins: Multitask signaling molecules linking fibroblast, endothelial and immune cell programs in the tumor microenvironment

“…Expression and secretion of Gal-3 has been well documented in NFs [46][47][48][49][50] and its silencing at the genetic level led to inhibition of fibroblast proliferation [51]. Similarly, Gal-3 knockout in mouse embryonic fibroblasts (MEFs) or Gal-3-depletion in human skin NFs decreased cell proliferation and increased premature senescence, highlighting a role for Gal-3 in senescence regulation [52].…”