Divergent Sp1 Protein Levels May Underlie Differential Expression of UDP-Glucose Dehydrogenase by Fibroblasts

Abstract

UDP-glucose dehydrogenase (UGDH) catalyzes the formation of UDP-glucuronate. Glucuronate represents an integral component of the glycosaminoglycan, hyaluronan, which accumulates in orbital Graves disease. Here we report that orbital fibroblasts express higher levels of UGDH than do those from skin. This is a consequence of greater UGDH gene promoter activity and more abundant steady-state UGDH mRNA. Six Sp1 sites located in the proximal 550 bp of the UGDH gene promoter appear to determine basal promoter activity, as does a previously unrecognized 49-bp sequence spanning ؊1436 nucleotides (nt) and ؊1388 nt that negatively affects activity. Nuclear Sp1 protein is more abundant in orbital fibroblasts, and its binding to specific sites on DNA is greater than that in dermal fibroblasts. Mutating each of these Sp1 sites in a UGDH gene promoter fragment, extending from ؊1387 to ؉71 nt and fused to a luciferase reporter, results in divergent activities when transfected in orbital and dermal fibroblasts. Reducing Sp1 attenuated UGDH gene promoter activity, lowered steady-state UGDH mRNA levels, and reduced UGDH enzyme activity. Targeting Sp1 and UGDH with specific siRNAs also lowered hyaluronan synthase-1 (HAS-1) and HAS-2 levels and reduced hyaluronan accumulation in orbital fibroblasts. These findings suggest that orbital fibroblasts express high levels of UGDH in an anatomic-specific manner, apparently the result of greater constitutive Sp1. These high UGDH levels may underlie susceptibility of the orbit to localized overproduction of hyaluronan in Graves disease.

Expression of Thyrotropin Receptor, Thyroglobulin, Sodium-Iodide Symporter, and Thyroperoxidase by Fibrocytes Depends on AIRE

“…Knockdown studies were conducted by transfecting fibrocytes with specific siRNA targeting AIRE. Transfections into fibrocytes, OF, and DF were conducted as described by Fernando et al (14) and Tsui et al (29), respectively. siRNA specifically targeting AIRE and its scrambled control were transfected into fibrocytes and fibroblasts as described previously (30).…”

TSH-receptor-expressing fibrocytes and thyroid-associated ophthalmopathy

“…This is associated with increased de novo adipogenesis, resulting in increased fat volume. Orbital fibroblasts express the molecular machinery necessary for HA synthesis, including UDP-glucose dehydrogenase 75,76 and the terminal hyaluronan synthases, of which three have been identified 77,78 . When activated by cytokines such as IL-1β, CD40 ligand, leukoregulin, prostaglandin D 2 , or growth factors such as IGF-1 and bTSH, fibroblasts express levels of these enzymes that result in accelerated HA production 33,79–82 .…”

Role of insulin-like growth factor-1 (IGF-1) pathway in the pathogenesis of Graves’ orbitopathy

“…Orbital fibroblasts express multiple enzymes involved in the biosynthesis of the glycosaminoglycan, hyaluronan. These include all three isozymes of hyaluronan synthase (18) and the upstream enzyme, UDP-glucose dehydrogenase (19,20). Orbital fibroblasts, especially those from patients with GO, represent a heterogeneous cell population that can be divided into subsets based on the display of the glycoprotein, Thy-1 (15-17).…”

“…Orbital fibroblasts in vitro produce remarkably large amounts of hyaluronan when activated by inflammatory cytokines such as IL-1β, interferon γ, leukoregulin, and CD154 (9,19,52,53). Moreover, they express all three hyaluronan synthase isozymes (HAS) (18) as well as extremely high levels of the enzyme, UDP glucose dehydrogenease (19,20). All of these enzymes are up-regulated by IL-1β, actions that are mediated at the pretranslational level.…”