Analysis of human hyaluronan synthase gene transcriptional regulation and downstream hyaluronan cell surface receptor mobility in myofibroblast differentiation.

Abstract

The ubiquitous extracellular glycosaminoglycan hyaluronan (HA) is a polymer composed of repeated disaccharide units of alternating D-glucuronic acid and D-N-acetylglucosamine residues linked via alternating β-1,4 and β-1,3 glycosidic bonds. Emerging data continue to reveal functions attributable to HA in a variety of physiological and pathological contexts. Defining the mechanisms regulating expression of the human hyaluronan synthase (HAS) genes that encode the corresponding HA-synthesizing HAS enzymes is therefore important in the context of HA biology in health and disease. We describe here methods to analyze transcriptional regulation of the HAS and HAS2-antisense RNA 1 genes. Elucidation of mechanisms of HA interaction with receptors such as the cell surface molecule CD44 is also key to understanding HA function. To this end, we provide protocols for fluorescent recovery after photobleaching analysis of CD44 membrane dynamics in the process of fibroblast to myofibroblast differentiation, a phenotypic transition that is common to the pathology of fibrosis of large organs such as the liver and kidney.