Integration of the Activation of the Human Hyaluronan Synthase 2 Gene Promoter by Common Cofactors of the Transcription Factors Retinoic Acid Receptor and Nuclear Factor κB

Abstract

Hyaluronan (HA) is a polysaccharide of the vertebrate extracellular matrix, produced by three related HA synthases (HASs) that influence numerous physiological processes. We screened the first 2250 bp of the HAS2 promoter for transcription factor response elements (REs) in silico and found 1 cluster of 2 retinoic acid (RA) REs, 3 discrete NF-B factors, and 12 Sp1 REs. In parallel, we scanned nine overlapping promoter regions in HaCaT human immortalized keratinocytes using chromatin immunoprecipitation assays to identify binding of mediator, coactivator, and corepressor proteins and Sp1 transcription factor in response to all-trans-RA and tumor necrosis factor-␣ (TNF-␣). We found that all-trans-RA modulated the binding of the RA receptor and several coregulators to the region containing the RARE cluster at position ؊1230. The importance of this region is supported in reporter gene assays by the all-trans-RA induction of the respective promoter region. Similarly, we showed by chromatin immunoprecipitation assays as well as by gel-shift assays with nuclear extracts that TNF-␣ induced NF-B binding to regions at positions ؊380, ؊1420, and ؊1890, demonstrated its association with RNA polymerase II and cofactor proteins, and confirmed the functionality of the respective promoter regions in vivo. These findings partially explain the induction of HAS2 mRNA by all-trans-RA and TNF-␣ and provide an example how the action of different transcription factor families can use the same cofactors.The glycosaminoglycan HA 2 is a high molecular mass linear non-sulfated polysaccharide that is a key component of the vertebrate extracellular matrix and has a variety of functions during and after development (1, 2). Its unique physiochemical properties and its interactions with specific cell surface receptors provide HA with a central role in cellular migration, adhesion, and proliferation (3). HA is synthesized by the enzymes HAS1, HAS2, and HAS3, located at the plasma membrane (4). Modified or aberrant HA metabolism is important in diseases such as malignancies, osteoarthritis, and pulmonary and vascular disorders along with other immune and inflammatory diseases (5). In the skin epidermis the narrow extracellular space surrounding keratinocytes contains a high concentration of HA (6). Keratinocyte growth and differentiation in both the normal and diseased epidermis are regulated by endocrine and paracrine signaling molecules such as the nuclear hormone alltrans-RA and epidermal growth factor (7). Direct evidence for the biological role of HA in epidermal keratinocytes emerged by the finding that HAS2-mediated HA synthesis controls the migration rate of keratinocytes in scratch-wounded monolayer cultures (8).It is known that a number of transcription factors such as RAR, signal transducers and activators of transcription 3, and Sp1 are involved in the regulation of HAS2 mRNA synthesis (7, 9); however, a systematic analysis of the promoter region has not been performed to compare the mechanisms of activation utilized by these transcri...

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.…”

“…Mannose and Glucosamine Regulate the Levels of HAS2 mRNA-Because the synthesis of hyaluronan showed an attenuated response to substrate supply, we measured the expression level of HAS2 mRNA, the dominant HAS isoenzyme in these cells (16). Quantitative RT-PCR indicated that enhanced HAS2 expression took place at 20 mM mannose already after 2 h, reached a 90% increase at 6 h, and decreased to 15% after 24 h (Fig.…”

“…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).…”