Hyaluronic acid regulates cellular UDP-GlcNAc levels through CD44 to affect glycosylation and cell biological functions

Hyaluronic acid (HA) is a key component of the extracellular matrix. Higher HA levels are strongly associated with poor prognosis in advanced cancer. Notably, the biosynthesis of N-glycans, O-GlcNAc, and HA all depend on UDP-GlcNAc as the essential donor substrate. Therefore, there may be functional relationships among different glycan types, although the specific mechanisms behind these interactions remain unclear. We established knockout (KO) cell lines for hyaluronan synthase 2 (HAS2) and CD44 in HeLa and PANC-1 cell lines, which express relatively high levels of HAS2. Results from cell proliferation, Transwell, wound-healing, and colony assays showed that proliferation, migration, and clonogenic capacity were significantly reduced in HAS2- or CD44-KO cells compared to wild-type cells. Lectin blot and HPLC analyses revealed increased levels of intracellular UDP-GlcNAc, O-GlcNAcylation, and GlcNAc-branched N-glycans in HAS2 KO cells. These changes were reversed by adding exogenous HA to HAS2 KO cells or by restoring HAS2 expression. Interestingly, HA effects were not observed in CD44 KO cells, indicating the key role of CD44 in mediating these HA-induced changes. Additionally, CD44 KO significantly reduced β-catenin levels and cell migration, which could be rescued with a β-catenin activator. Our findings suggest that cells sense extracellular HA levels through CD44 to induce CD44-dependent β-catenin signaling, potentially regulating fructose-6-phosphate amidotransferase, a rate-limiting enzyme in the hexosamine biosynthetic pathway responsible for the synthesis of UDP-GlcNAc. These results provide a potential mechanistic connection between extracellular HA and intracellular glycosylation, offering new insights into the diverse roles of HA in cell biology.