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.
Highlights
UDP-glucose dehydrogenase (UGDH,3 EC1.1.1.22) catalyzes the conversion of UDP-D-glucose to UDP-D-glucuronate (UDP-GlcUA) [1] as defined by the chemical reaction
Using a polyclonal anti-human UGDH Ab directed at the carboxyl terminus, Western blot analysis revealed a single 55-kDa band expressed under basal culture conditions in several orbital fibroblast strains, each derived from a different donor (Fig. 1A)
UGDH is widely expressed in nature, and its function and regulation have been characterized in bacteria [40, 41]
Summary
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. Thyroid-associated ophthalmopathy (TAO) is a manifestation of GD where orbital tissue accumulates hyaluronidase-sensitive glycosaminoglycan and becomes infiltrated by mast cells, and T and B lymphocytes [21,22,23] The cytokines they generate activate orbital fibroblasts to produce excessive hyaluronan, which leads to tissue expansion and proptosis [24]. We find that levels of nuclear Sp1 protein and binding to 6 Sp1 recognition sites in the gene promoter are considerably greater in orbital fibroblasts. This enzyme may underlie, at least in part, the peculiar phenotype of orbital fibroblasts that renders the orbit susceptible to TAO
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