O-GlcNAc-Mediated Regulation of Galectin Expression and Secretion in Human Promyelocytic HL-60 Cells Undergoing Neutrophilic Differentiation

Abstract

In this study, we have tested the hypothesis that the expression and secretion of galectins are driven through mechanisms globally impacted by homeostatic regulation involving the post-translational modification of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc). We showed that neutrophilic differentiation of HL-60 cells induced by all-trans retinoic acid (ATRA) and 6-diazo-5-oxo-L-norleucine (DON) was associated with a significant drop of cellular O-GlcNAc levels in serum-contained and serum-free cell culture media. Galectin gene and protein expression profiles in HL-60 cells were specifically modified by ATRA and by inhibitors of O-GlcNAc cycle enzymes, however overall trends for each drug were similar between cells growing in the presence or absence of serum except for LGALS9 and LGALS12. The secretion of four galectins (-1, -3, -9, and -10) by HL-60 cells in a serum-free medium was stimulated by O-GlcNAc-reducing ATRA and DON while O-GlcNAc-elevating thiamet G (O-GlcNAcase inhibitor) failed to change the basal levels of extracellular galectins. Taken together, these results demonstrate that O-GlcNAc homeostasis is essential not only for regulation of galectin expression in cells but also for the secretion of multiple members of this protein family, which can be an important novel aspect of unconventional secretion mechanisms.

Galectin-9 interacts with Vamp-3 to regulate cytokine secretion in dendritic cells

“…On the contrary, no cytokine could be detected at the Golgi complex of LPS-treated WT cells, which aligns with previous publications showing that in DCs and macrophages cytokine accumulation at the Golgi cannot be observed 2–4 h after LPS stimulation [ 11 , 12 , 52 ]. Furthermore, galectin-9 has been reported to be secreted to the extracellular space in many cells [ 53 – 55 ]and it is thus plausible that in DCs galectin-9 is also secreted together with cytokines as a functional complex in addition to facilitating their intracellular trafficking.…”

Insights into cAMP‐dependent molecular mechanisms regulating expression and function of LGALS16 gene in choriocarcinoma JEG‐3 cells

“…To prepare protein lysates, cell monolayers in six‐well plates were rinsed twice with DPBS, and then 300 µL RIPA buffer per well were added for 10 min with gentle pipetting of the lysate several times. All subsequent steps were performed as described earlier (McTague et al, 2022), with the exception that the ready‐to‐use 10% Mini‐PROTEAN TGX gels (#4561034; Bio‐Rad) were used for sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and Luminata™ Forte Western HRP substrate (#WBLUF0100) from Millipore was used for developing western blots. Primary antibodies included rabbit anti‐hCG beta polyclonal antibody from Thermo Fisher (#PA5‐16265), rabbit anti‐galectin‐13/16 monoclonal antibody from Abcam (#ab272716), and mouse anti‐β‐actin monoclonal antibody from Santa Cruz (#sc‐47778).…”

Expression and secretion of galectin‐12 in the context of neutrophilic differentiation of human promyeloblastic HL‐60 cells

“…Galectins are a family of soluble proteins initially characterized by their binding affinity towards β-galactoside-containing glycans (Johannes et al, 2018). Galectin-12 is a tissue-specific galectin that has previously been shown to play a role in the differentiation and/or polarization of various cell types including adipocytes, sebocytes, macrophages, and neutrophils (Harrison et al, 2007;McTague et al, 2022;Tsao et al, 2022;Wan et al, 2016;Xue et al, 2016;Yang et al, 2004). It is well-established that galectin-12 is required as a lipogenic factor for the proper differentiation of adipocytes and sebocytes engaging signaling pathways associated with lipogenesis in human, mouse, and porcine models (Wu et al, 2018;Yang et al, 2004Yang et al, , 2011.…”

“…is the only galectin tested in HL-60 cells thus far, that had inhibition of secretion upon neutrophilic differentiation(McTague et al, 2022).F I G U R E 5 A proposed role and regulation of galectin-12 in neutrophilic differentiation of HL-60 cells.LGALS12 is proposed to serve as a marker of N1 and N2 neutrophilic polarization due to its varied expression between two distinct phenotypes of neutrophil-like HL-60 cells induced by ATRA and DMSO. Both phenotypes accumulate lipid droplets (LD), which inhibit the secretion of galectin-12.…”

“…Further Gene Set Enrichment Analysis (GSEA) based on Biological Process GO demonstrated that terms associated with cell division, DNA replication and repair, and translation and protein folding were found to be significantly downregulated with DMSO but not ATRA.Meanwhile, terms associated with cell signal reception and transduction, and cell cytoskeletal functions were upregulated to a greater extent by ATRA compared to DMSO (Figure1d, Supporting Information S1: FigureS2). Thus, ATRA-and DMSO-induced neutrophilic phenotypes of HL-60 cells are characterized by significant differences in gene expression profiles.3.2 | Opposing changes in the expression ofLGALS12 in HL-60 cells treated with ATRA and DMSO Following the bioinformatics analysis above and discrepancy of reports on the expression and functions of LGALS12 in myeloid cells(McTague et al, 2022;Vinnai et al, 2017;Xue et al, 2016), we sought to revisit this aspect in a model of neutrophilic differentiation of HL-60 cells. The cells were treated for 3 days with ATRA (1 μM) and DMSO (1.3%) and the following features of differentiated cells were verified: nuclear morphology, expression of differentiation markers, and functional responses to phagocyte membrane NADPH oxidase inducers such as fMLP (an agonist of formyl peptide receptors) and…”