A 2D and 3D cell culture protocol to study O-GlcNAc in sphingosine-1-phosphate mediated fibroblast contraction.

O-linked ß-N-acetylglucosamine (O-GlcNAc) is an abundant posttranslational modification involved in a wide range of signaling pathways, but its specific role in regulating biological processes remains unclear. This protocol describes approaches to understand O-GlcNAc's role in fibroblast contraction. Specifically, cellular O-GlcNAc levels are controlled through treatment of fibroblasts with inhibitors in both 2D and 3D cultures. We then describe 2D contraction assay and 3D collagen gel contraction assay to analyze the effect of the modification on sphingosine-1-phosphate signaling for contraction. For complete details on the use and execution of this protocol, please refer to Pedowitz et al. (2021).

O-GlcNAc modification of MYPT1 modulates lysophosphatidic acid-induced cell contraction in fibroblasts.

Thousands of proteins have been found to be modified by O-GlcNAc, a common glycosylation modification of serine and threonine residues throughout the cytosol and nucleus. O-GlcNAc is enzymatically added and removed from proteins, making it a potential dynamic regulator of cell signaling. However, compared with other posttranslational modifications like phosphorylation, relatively few O-GlcNAc-regulated pathways have been discovered and biochemically characterized. We previously discovered one such pathway, where O-GlcNAc controls the contraction of fibroblasts initiated by the signaling lipid sphingosine-1-phosphate. Specifically, we found that O-GlcNAc modification of the phosphatase MYPT1 maintains its activity, resulting in dephosphorylation and deactivation of the myosin light chain of the actinomyosin complex. Another signaling lipid that leads to contraction of fibroblasts is lysophosphatidic acid, and this signaling pathway also converges on MYPT1 and actinomyosin. We therefore rationalized that O-GlcNAc would also control this pathway. Here, we used a combination of small molecule inhibitors, 2D and 3D cell cultures, and biochemistry to confirm our hypothesis. Specifically, we found that O-GlcNAc levels control the sensitivity of mouse and primary human dermal fibroblasts to lysophosphatidic acid-induced contraction in culture and the phosphorylation of MLC and that MYPT1 O-GlcNAc modification is responsible. These findings further solidify the importance of O-GlcNAc in regulating the biology of fibroblasts in response to procontractile stimuli.