Department of Bioengineering, George Mason University, Manassas, Virginia, United States of America.
Institute of Biomedical Engineering, Polytechnique Montréal, Montréal, QC, Canada.
PLoS One. 2022 Sep 15;17(9):e0274420. doi: 10.1371/journal.pone.0274420. eCollection 2022.
UDP-glucose dehydrogenase (UGDH) generates essential precursors of hyaluronic acid (HA) synthesis, however mechanisms regulating its activity are unclear. We used enzyme histostaining and quantitative image analysis to test whether cytokines that stimulate HA synthesis upregulate UGDH activity. Fibroblast-like synoviocytes (FLS, from N = 6 human donors with knee pain) were cultured, freeze-thawed, and incubated for 1 hour with UDP-glucose, NAD+ and nitroblue tetrazolium (NBT) which allows UGDH to generate NADH, and NADH to reduce NBT to a blue stain. Compared to serum-free medium, FLS treated with PDGF showed 3-fold higher UGDH activity and 6-fold higher HA release, but IL-1beta/TGF-beta1 induced 27-fold higher HA release without enhancing UGDH activity. In selected proliferating cells, UGDH activity was lost in the cytosol, but preserved in the nucleus. Cell-free assays led us to discover that diaphorase, a cytosolic enzyme, or glutathione reductase, a nuclear enzyme, was necessary and sufficient for NADH to reduce NBT to a blue formazan dye in a 1-hour timeframe. Primary synovial fibroblasts and transformed A549 fibroblasts showed constitutive diaphorase/GR staining activity that varied according to supplied NADH levels, with relatively stronger UGDH and diaphorase activity in A549 cells. Unilateral knee injury in New Zealand White rabbits (N = 3) stimulated a coordinated increase in synovial membrane UGDH and diaphorase activity, but higher synovial fluid HA in only 2 out of 3 injured joints. UGDH activity (but not diaphorase) was abolished by N-ethyl maleimide, and inhibited by peroxide or UDP-xylose. Our results do not support the hypothesis that UGDH is a rate-liming enzyme for HA synthesis under catabolic inflammatory conditions that can oxidize and inactivate the UGDH active site cysteine. Our novel data suggest a model where UGDH activity is controlled by a redox switch, where intracellular peroxide inactivates, and high glutathione and diaphorase promote UGDH activity by maintaining the active site cysteine in a reduced state, and by recycling NAD+ from NADH.
UDP-葡萄糖脱氢酶（UGDH）生成透明质酸（HA）合成的必需前体，但调节其活性的机制尚不清楚。我们使用酶组织化学染色和定量图像分析来测试刺激 HA 合成的细胞因子是否上调 UGDH 活性。纤维母细胞样滑膜细胞（FLS，来自 N = 6 名膝关节疼痛的人类供体）进行培养、冻融，并在含有 UDP-葡萄糖、NAD+和硝基蓝四唑（NBT）的培养基中孵育 1 小时，使 UGDH 生成 NADH，NADH 将 NBT 还原为蓝色染色剂。与无血清培养基相比，PDGF 处理的 FLS 显示 UGDH 活性增加 3 倍，HA 释放增加 6 倍，但 IL-1β/TGF-β1 诱导 HA 释放增加 27 倍，而不增强 UGDH 活性。在选定的增殖细胞中，UGDH 活性在细胞质中丢失，但在核中保留。细胞游离测定使我们发现，细胞色素 b2 还原酶（一种细胞质酶）或谷胱甘肽还原酶（一种核酶）是 NADH 在 1 小时内将 NBT 还原为蓝色甲臜染料所必需且充分的。原代滑膜成纤维细胞和转化的 A549 成纤维细胞显示出组成型二氢乳清酸酶/GR 染色活性，该活性根据提供的 NADH 水平而变化，A549 细胞中的 UGDH 和二氢乳清酸酶活性相对较强。新西兰白兔单侧膝关节损伤（N = 3）刺激滑膜膜 UGDH 和二氢乳清酸酶活性协同增加，但只有 3 个受伤关节中的 2 个关节滑液中 HA 升高。N-乙基马来酰亚胺可消除 UGDH 活性（但不消除二氢乳清酸酶），而过氧化物或 UDP-木糖可抑制 UGDH 活性。我们的结果不支持 UGDH 是在可以氧化和失活 UGDH 活性位点半胱氨酸的分解代谢炎症条件下控制 HA 合成的限速酶的假说。我们的新数据表明，UGDH 活性受氧化还原开关控制，细胞内过氧化物使 UGDH 失活，高谷胱甘肽和二氢乳清酸酶通过维持活性位点半胱氨酸处于还原状态并从 NADH 循环 NAD+来促进 UGDH 活性。
