The Human Hyaluronan Synthase 2 Gene Is a Primary Retinoic Acid and Epidermal Growth Factor Responding Gene

Abstract

Hyaluronan is an abundant and rapidly turned over matrix molecule between the vital cell layers of the epidermis and subject to large concentration changes associated with keratinocyte proliferation, migration, and differentiation induced by paracrine and endocrine factors like epidermal growth factor (EGF) and all-transretinoic acid (RA). We found that in REK cells EGF and all-trans-RA up-regulated hyaluronan synthase 2 (Has2) gene expression within 2 h 4-fold each and in HaCaT human immortal keratinocytes 8-and 33-fold, respectively. The first 10 kb of the human Has2 promoter were scanned in silico and in vitro for potential response elements of signal transducer and activator of transcription (STAT) or RA receptor (RAR) proteins. We identified a STAT-response element in the proximal promoter region and confirmed its functionality in response to EGF by chromatin immunoprecipitation (ChIP) assays. Direct in vitro binding of RARs to four RARE candidates within the Has2 promoter could not be observed at stringent gel shift conditions, but reporter gene assays demonstrated functionality of a complex of two of these RAREs located ϳ1200 bp upstream of the transcription start site. Moreover, ChIP assays using antibodies against nine nuclear proteins monitored all-trans-RAdependent binding of RAR, retinoid X receptor, mediator protein, and RNA polymerase II and also histone 4 acetylation to a promoter region containing the complex RARE. Taken together, the human Has2 gene is a potent primary EGF and all-trans-RA responding gene with a complex regulation.The glycosaminoglycan hyaluronan is a high molecular mass polysaccharide that is a key component of the vertebrate extracellular matrix and is involved in a wide range of cellular functions including migration, adhesion, and proliferation by its unique physicochemical properties and interactions with specific cell surface receptors (1). Hyaluronan is synthesized by the Has enzymes Has1, Has2, and Has3 at the plasma membrane (2). In skin epidermis, the narrow extracellular space surrounding keratinocytes contains a high concentration of hyaluronan, but it is found mainly between the basal and spinous cell layers of normal human epidermis and much less in terminally differentiated layers (3). Both in normal and diseased epidermis, keratinocyte growth and differentiation are regulated by paracrine and endocrine signaling molecules, such as EGF 1 and the nuclear hormone all-trans-RA. Interestingly, hyaluronan synthesis rate is stimulated by EGF in epidermal keratinocytes in monolayer (4) and organotypic cultures (5) and by all-trans-RA in human skin organ cultures (3). Direct evidence for the biological role of hyaluronan in epidermal keratinocytes emerged by the finding that Has2-mediated hyaluronan synthesis controls the migration rate of keratinocytes in scratch-wounded monolayer cultures (6). Hyaluronan concentration is closely correlated with the proliferative activity and volume of the vital part of the epidermis and inversely related with the markers of differentiat...

Cellular Content of UDP-N-acetylhexosamines Controls Hyaluronan Synthase 2 Expression and Correlates with O-Linked N-Acetylglucosamine Modification of Transcription Factors YY1 and SP1

“…Several functional REs on the HAS2 promoter have been verified previously (7,11,16). However, following 6-h treatments with mannose and glucosamine, no changes in the recruitment of CREB1, NF-B, and STAT3 were noted on the first 2250 bp of the promoter (data not shown), suggesting that the previously described signals related to G-protein-coupled receptors, inflammation or cell survival, and growth factors like EGF (7,11,16) are not part of the pathways that carry UDP-HexNAc information to HAS2 transcription.…”

“…We have previously characterized the NF-B, STAT3, and SP1 sites on the HAS2 promoter (7,16). The CREB1-and YY1-binding sites on the HAS2 promoter were identified using the net-based program ConSite applying a transcription factor binding search cutoff of 85% and our own software for modified hexamer-binding site searching (retinoid acid receptor REs) (29).…”

“…In keratinocyte cultures, EGF, keratinocyte growth factor, TNF␣, and retinoic acid induce, whereas TGF␤ inhibits, HAS2 expression (8, 10, 13, 16). Accordingly, the HAS2 promoter has been shown to contain functional response elements (REs) 3 for different transcription factors, including retinoid acid receptor, NF-B, CREB1 (cAMP response elementbinding protein 1), and SP1 (specificity protein 1) (7,11,16).Besides by the protein expression of hyaluronan synthase (HAS) enzymes, hyaluronan synthesis is also controlled by the availability of the hyaluronan precursors, the substrates of HAS. Raising cellular UDP-GlcUA content stimulates hyaluronan synthesis, whereas a low concentration of UDP-GlcUA can limit the synthesis (12, 17).…”

“…In keratinocyte cultures, EGF, keratinocyte growth factor, TNF␣, and retinoic acid induce, whereas TGF␤ inhibits, HAS2 expression (8, 10, 13, 16). Accordingly, the HAS2 promoter has been shown to contain functional response elements (REs) 3 for different transcription factors, including retinoid acid receptor, NF-B, CREB1 (cAMP response elementbinding protein 1), and SP1 (specificity protein 1) (7,11,16).…”

“…Changes in hyaluronan production have been associated mostly with the expression level of HAS genes (3-6), especially in keratinocytes (7)(8)(9)(10)(11)(12)(13). Of the three genes, particularly HAS2 is subject to regulation by growth factors, cytokines, and hormones (4,14,15).…”