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...

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...

The candidate human tumor suppressor gene cyclin C is a primary target of the anti-proliferative hormone 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], but binding sites for the 1α,25(OH)2D3 receptor (VDR), so-called 1α,25(OH)2D3 response elements (VDREs), have not yet been identified in the promoter of this gene. We screened various cancer cell lines by quantitative PCR and found that the 1α,25(OH)2D3 inducibility of cyclin C mRNA expression, in relationship with the 24-hydroxylase (CYP24) gene, was best in MCF-7 human breast cancer cells. To characterize the molecular mechanisms, we analyzed 8.4 kb of the cyclin C promoter by using chromatin immunoprecipitation assays (ChIP) with antibodies against acetylated histone 4, VDR and its partner receptor, retinoid X receptor (RXR). The histone 4 acetylation status of all 23 investigated regions of the cyclin C promoter did not change significantly in response to 1α,25(OH)2D3, but four independent promoter regions showed a consistent, 1α,25(OH)2D3-dependent association with VDR and RXR over a time period of 240 min. Combined in silico/in vitro screening identified in each of these promoter regions a VDRE and reporter gene assays confirmed their functionality. Moreover, re-ChIP assays monitored simultaneous association of VDR with RXR, coactivator, mediator and RNA polymerase II proteins on these regions. Since cyclin C protein is associated with those mediator complexes that display transcriptional repressive properties, this study contributes to the understanding of the downregulation of a number of secondary 1α,25(OH)2D3-responding genes.