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.

CD44/CD44v6 a Reliable Companion in Cancer-Initiating Cell Maintenance and Tumor Progression

“…However, the major transcription factors engaged in HAS1,−2, and−3 transcription are not defined. Second, more information on HA degradation by Hyal is required, amongst others the regulation of Hyal transcription (Negi et al, 2012; Midgley and Bowen, 2015). Taking into account that Hyal and low MW HA improve drug efficacy (Bourguignon et al, 2009b), and that HA-CD44 cross-linking regulates drug transporter expression (Misra et al, 2005; Kathawala et al, 2015), answers are urgently awaited (Sebens and Schafer, 2012; Lokeshwar et al, 2014).…”

CD44, Hyaluronan, the Hematopoietic Stem Cell, and Leukemia-Initiating Cells

“…However, this as well as the major transcription factors engaged in HAS1, -2, and -3 transcription remain to be defined. Transcriptional regulation of hyaluronidases also awaits unraveling ( 320 , 321 ). Taking into account that hyaluronidase as well as small HA oligosaccharides can improve drug efficacy ( 318 ), and that HA-CD44 cross-linking regulates expression of drug transporters ( 315 , 316 ), filling this gap becomes demanding to improve therapeutic targeting of HA [review in Ref.…”

Regulation of Synthesis and Roles of Hyaluronan in Peritoneal Dialysis

“…To locate the HAS2 promoter, we carried out HAS2-specific 5′-rapid amplification of cDNA ends (5′RACE) on polyadenylated RNA extracted from renal proximal tubular epithelial cells and located the TSS 0.130 kb upstream of the 5′ end of HAS2 reference mRNA sequence NM_005328 [ 45 ]. We then generated luciferase reporter vectors bearing nested fragments spanning the first 0.8 kb upstream of this new TSS [ 45 , 46 ]. Luciferase analysis showed consistent promoter activity mediated by a minimum-sized fragment of 0.121 kb, within which we identified promoter sequences conserved in selected mammals [ 45 , 46 ].…”

“…We then generated luciferase reporter vectors bearing nested fragments spanning the first 0.8 kb upstream of this new TSS [ 45 , 46 ]. Luciferase analysis showed consistent promoter activity mediated by a minimum-sized fragment of 0.121 kb, within which we identified promoter sequences conserved in selected mammals [ 45 , 46 ]. Similar methods have recently been used to identify the human HAS3 promoter [ 47 ].…”

“…We have also identified the HAS1 TSS, adding a further 26 nucleotides to reference sequence NM_001523, and analysed the upstream HAS1 promoter region in renal proximal tubular epithelial cells [ 46 , 53 ], but a full characterisation of factors regulating HAS expression in HPMCs has not been carried out. In addition, little is known about HPMC expression of long noncoding RNAs (including HAS2-AS1) and of microRNAs, both of which are highly likely to regulate HAS expression.…”