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-trans-retinoic 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 ∼1200 bp upstream of the transcription start site. Moreover, ChIP assays using antibodies against nine nuclear proteins monitored all-trans-RA-dependent 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. 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-trans-retinoic 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 ∼1200 bp upstream of the transcription start site. Moreover, ChIP assays using antibodies against nine nuclear proteins monitored all-trans-RA-dependent 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 (1Tammi M.I. Day A.J. Turley E.A. J. Biol. Chem. 2002; 277: 4581-4584Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar). Hyaluronan is synthesized by the Has enzymes Has1, Has2, and Has3 at the plasma membrane (2Weigel P.H. Hascall V.C. Tammi M. J. Biol. Chem. 1997; 272: 13997-14000Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar). 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 (3Tammi R. Ripellino J.A. Margolis R.U. Maibach H.I. Tammi M. J. Investig. Dermatol. 1989; 92: 326-332Abstract Full Text PDF PubMed Scopus (128) Google Scholar). Both in normal and diseased epidermis, keratinocyte growth and differentiation are regulated by paracrine and endocrine signaling molecules, such as EGF 1The abbreviations used are: EGF, epidermal growth factor; ARP0, acidic riboprotein P0; CBP, CREB-binding protein; ChIP, chromatin immunoprecipitation; DR5, direct repeat spaced by 5 nucleotides; EGFR, epidermal growth factor receptor; FBS, fetal bovine serum; HABC, hyaluronan binding complex; Has, hyaluronan synthase; MED, mediator; RE, response element; Pol II, RNA polymerase II; pPol II, phosphorylated RNA polymerase II; pSTAT, phosphorylated STAT; RA, retinoic acid; RAR, retinoic acid receptor; RARE, retinoic acid response element; REK, rat epidermal keratinocyte; RXR, retinoid X receptor; STAT, signal transducer and activator of transcription; TSS, transcription start site; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline. and the nuclear hormone all-trans-RA. Interestingly, hyaluronan synthesis rate is stimulated by EGF in epidermal keratinocytes in monolayer (4Pienimäki J.P. Rilla K. Fulop C. Sironen R.K. Karvinen S. Pasonen S. Lammi M.J. Tammi R. Hascall V.C. Tammi M.I. J. Biol. Chem. 2001; 276: 20428-20435Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar) and organotypic cultures (5Pasonen-Seppänen S. Karvinen S. Törrönen K. Hyttinen J.M. Jokela T. Lammi M.J. Tammi M.I. Tammi R. J. Investig. Dermatol. 2003; 120: 1038-1044Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) and by all-trans-RA in human skin organ cultures (3Tammi R. Ripellino J.A. Margolis R.U. Maibach H.I. Tammi M. J. Investig. Dermatol. 1989; 92: 326-332Abstract Full Text PDF PubMed Scopus (128) Google Scholar). 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 (6Rilla K. Lammi M.J. Sironen R. Törrönen K. Luukkonen M. Hascall V.C. Midura R.J. Hyttinen M. Pelkonen J. Tammi M. Tammi R. J. Cell Sci. 2002; 115: 3633-3643Crossref PubMed Scopus (59) Google Scholar). 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 differentiation, suggesting that hyaluronan synthesis regulated by Has2 and Has3 is an important component in the proliferative reactions of the epidermis and is also involved in the epidermal differentiation process (5Pasonen-Seppänen S. Karvinen S. Törrönen K. Hyttinen J.M. Jokela T. Lammi M.J. Tammi M.I. Tammi R. J. Investig. Dermatol. 2003; 120: 1038-1044Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). EGF is one of the most powerful molecules influencing the behavior of keratinocytes (7Piepkorn M. Pittelkow M.R. Cook P.W. J. Investig. Dermatol. 1998; 111: 715-721Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). The growth factor binds to the EGF receptor (EGFR), which belongs to the erbB receptor tyrosine kinase family (8Holbro T. Hynes N.E. Annu. Rev. Pharmacol. Toxicol. 2004; 44: 195-217Crossref PubMed Scopus (507) Google Scholar). Activated EGFRs dimerize, which promotes autophosphorylation of specific tyrosines located in their cytoplasmic domain. These phosphorylated residues serve as docking sites for a variety of signaling molecules, whose recruitment finally stimulates numerous transcription factors including members of the STAT protein family (9Grandis J.R. Drenning S.D. Chakraborty A. Zhou M.Y. Zeng Q. Pitt A.S. Tweardy D.J. J. Clin. Investig. 1998; 102: 1385-1392Crossref PubMed Google Scholar). In this way, activated EGFRs induce via the tyrosine kinase Src the phosphorylation of STAT proteins 1, and 5 T. M. J. PubMed Scopus Google which to the STAT within promoter of of the with a for 1997; 277: PubMed Scopus Google Scholar). most members of the nuclear receptor the and two that a J. 2002; PubMed Scopus Google Scholar) and a Cell Biol. 1998; PubMed Scopus Google Scholar). binding a within the to interactions with and mediator proteins PubMed Google Scholar). the RARs to enzymes histone such as CREB-binding protein that chromatin In a RARs with a complex that as a to the basal transcription RNA polymerase II as its C. M. PubMed Scopus Google Scholar). In this RARs two chromatin and RARs to with the or or The a but the in epidermis is J. PubMed Scopus Google Scholar). RARs with the retinoid X receptor nuclear receptor family which also D.J. Full Text PDF PubMed Scopus Google Scholar). RAREs are by a direct repeat of two spaced by primary RA gene at one RARE in its promoter region and these RAREs are to be located to the transcription start of these A. PubMed Scopus Google Scholar). The protein of chromatin are the of these in acetylation and at residues of their the of the and their for the in the promoter a high of the chromatin is and the associated is for with protein involved in gene regulation. the of these a large chromatin and of transcription factor binding sites T. 2001; PubMed Scopus Google Scholar). for the direct of transcription via EGF or all-trans-RA is the of activated STAT proteins or to the basal or RAREs be located within activated chromatin in the promoter of EGF and RA The proximal promoter region of Has of which Has2 the basal J. J. M. T. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google the Has2 gene to be the for the hyaluronan synthesis In this observed that all-trans-RA up-regulated hyaluronan as as EGF in organotypic cultures REK cells and that Has2 gene expression rapidly in monolayer Has2 and hyaluronan were up-regulated in HaCaT human immortal In silico of the first bp of the human Has2 promoter the of binding sites of transcription factors and J. J. M. T. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google but the promoter is in this a in vitro of the first 10 kb of the human Has2 promoter and identified an in the proximal ChIP assays a of promoter of the human Has2 promoter all-trans-RA-dependent histone 4 acetylation and binding of RAR, RXR, protein, and Pol II to a promoter region containing a complex RARE. The human Has2 gene is a potent primary EGF and RA responding gene with a complex regulation. Cell REK cell the epidermis of rat in containing fetal bovine and 4 S. M. M. A. Tammi M. Tammi R. J. Investig. Dermatol. 2001; Full Text Full Text PDF PubMed Google Scholar) and the human keratinocyte HaCaT cell J. A. N.E. J. Cell Biol. PubMed Scopus Google Scholar) in with and 2 organotypic REK cells were at the rat as S. M. M. A. Tammi M. Tammi R. J. Investig. Dermatol. 2001; Full Text Full Text PDF PubMed Google Scholar). The organotypic REK cell cultures were for 2 with the for the first and 2 The of the cultures with the the cells were to the and for 10 the cultures were in in and with and to or ChIP, REK and HaCaT cells were at a of for with all-trans-RA EGF and kinase or in Hyaluronan of the keratinocyte cultures with a M. A. K. J. M. M. Tammi R. Tammi M. 2002; Google Scholar). The were with with and with bovine in the as (5Pasonen-Seppänen S. Karvinen S. Törrönen K. Hyttinen J.M. Jokela T. Lammi M.J. Tammi M.I. Tammi R. J. Investig. Dermatol. 2003; 120: 1038-1044Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). were by with the hyaluronan binding complex of the for 2 h at with in and with bovine for h at The as R. Tammi M.I. PubMed Scopus Google Scholar). The of hyaluronan and the were to the h at the were with with for and with The using a complex in and in The at the with 2 RNA and RNA and were using and respectively. of used as a in a synthesis using of in the of The for h at in an using the The were using the is the is and is the at which the is and specific are in monitored using at the of the and for of human and rat Has gene and the at at at at at at at at at at at at bp in a vitro and proteins were by in vitro using their expression M. A. C. PubMed Scopus Google Scholar) and as by the were by in the of The specific concentration of the receptor proteins to to to the of residues protein shift assays were with 10 of the in vitro proteins or of protein The proteins were for in a volume of of binding 10 of for were and for at were by 5 or in and cells were and in with of an RARE and the RARE of RAREs and of the Has2 promoter were with the kinase promoter the reporter containing were by these reporter and an expression for human M. A. C. PubMed Scopus Google Scholar) with 10 for at in a volume of with of the were to the with of 4 h this all-trans-RA or were also The cells were h of using the reporter gene and the signal reporter gene as by the The The were with to protein and factors were as the of activity of cells to that of ChIP proteins were to by to the to a concentration of for at with by to a concentration of and for 5 at a The and the cells were with The cells were by with a the cell were in 10 and the were to in of bp in by and the were in ChIP were with 5 of the antibodies at 4 with The against histone 4 antibodies against phosphorylated Pol II phosphorylated Pol II II, and were The were with of for 2 h at 4 with The were by for at 4 at and for 5 a with of the 2 high 2 and 10 the were with of 10 The were by of and for at with the and the by to a concentration of and at Full Text Full Text PDF PubMed Scopus Google Scholar). The proteins were by concentration and for h at The by and with volume of and 2 of using as a were used as a for with the 5 at and of at at specific and at with one for 10 at The and the of the chromatin are in The were by with of the specific for human Has2 promoter to to to to to to in a and EGF and Hyaluronan cultures of REK cells within 10 in a vital epidermis and layers of differentiated which were for the with EGF, or the of of vital in a of layers 1, In with (5Pasonen-Seppänen S. Karvinen S. Törrönen K. Hyttinen J.M. Jokela T. Lammi M.J. Tammi M.I. Tammi R. J. Investig. Dermatol. 2003; 120: 1038-1044Abstract Full Text Full Text PDF PubMed Scopus (151) Google S. M. M. A. Tammi M. Tammi R. J. Investig. Dermatol. 2001; Full Text Full Text PDF PubMed Google the epidermal and differentiation were associated with hyaluronan in the and cultures The kinase a the of the epidermis it the epidermal The of hyaluronan closely to epidermal cultures and a of the hyaluronan induced by all-trans-RA Has2 in REK by and the Has2 is a of hyaluronan in the epidermis (4Pienimäki J.P. Rilla K. Fulop C. Sironen R.K. Karvinen S. Pasonen S. Lammi M.J. Tammi R. Hascall V.C. Tammi M.I. J. Biol. Chem. 2001; 276: 20428-20435Abstract Full Text Full Text PDF PubMed Scopus (166) Google by the expression of the Has2 gene in response to the of as in the organotypic were in the primary of the Has2 the 2 h and the REK cells were as these in and conditions, found that the Has2 in REK cell correlated with the hyaluronan in organotypic REK cell to the expression of the gene acidic riboprotein also as all-trans-RA and EGF stimulated the Has2 and the of in a in a but not of Has2 the with all-trans-RA and a of the all-trans-RA response of the and EGF Has2 in HaCaT to a human and Has2 expression to the of in HaCaT human immortal keratinocytes Interestingly, in this cellular a in REK cell and EGF stimulated human Has2 and 33-fold, 2 h of and the in a In Has2 expression to of could be stimulated by but the by a with all-trans-RA and with of the RA of Has2 demonstrated a of 5 h but h not The Has2 or correlated with the of hyaluronan in of HaCaT cells a and EGF hyaluronan and and the in a observed the of Has2 hyaluronan to of within h and in the of all-trans-RA The between Has2 and hyaluronan be related to the of EGF and RA that the of be the of Has2 gene expression by all-trans-RA. In in rat and human keratinocytes all-trans-RA and EGF a of Has2 and hyaluronan The of an or Has2 signaling the basal Has2 and hyaluronan synthesis in human but not the of Has2 by all-trans-RA the of the in the of In to the human the Has2 of REK cells in monolayer to for STAT and within the Has2 response of the Has2 gene to all-trans-RA and EGF 2 h that the gene is a primary retinoid and EGF responding gene and that its promoter binding sites for RARs and STAT proteins. in silico of the first 10 kb of the human Has2 promoter for nuclear receptor binding of the in a the this to one identified four between two and between a of the STAT also found four We gel shift assays with these four using STAT protein stringent conditions, to gel shift A. C. J. PubMed Scopus Google M. C. J. Biol. PubMed Scopus Google and in to an the first RE, located at to upstream of the TSS, a binding of STAT The binding of in vitro and or in the four RAREs in to an RARE the RARE demonstrated a complex of the four could binding of or be The with and nuclear HaCaT cells not RAREs and 2 are of the complex RE, but with this containing nuclear receptor binding were not to complex in vitro not the RARE to the kinase it a all-trans-RA-dependent of reporter gene which is with that of the RARE in the promoter The of the rat Has2 promoter is not but it is that the human and rat gene are In of the transcription factor binding sites are between the two to an of the human to Taken together, in vitro for STAT protein and retinoid receptor binding sites within the human Has2 promoter in one within the proximal promoter and a RARE located ∼1200 bp upstream of the of RARs and STAT to the Has2 retinoid receptors and STAT proteins in cells with the human Has2 ChIP assays with all-trans-RA and HaCaT this for the of the human Has2 promoter that the RAREs and 1, and in In one region as for the of the the of in the J. Chem. PubMed Scopus Google Scholar) to in the Has2 promoter HaCaT cells that were in the of FBS, stimulated for with all-trans-RA or EGF, and for in the of ChIP assays were using antibodies against and Cell chromatin and were at with In the of all-trans-RA of the promoter associated with but region a of The of HaCaT cells with all-trans-RA for not in a of to 1, and and a binding of the receptor to region but also in an of and to region In retinoid also induced binding of and to 2 and and an of to region 5 and to region In the promoter region 4 with of the proteins. HaCaT cells with and EGF confirmed the of to region EGF The Has2 promoter STAT in the of growth factor EGF the of to region and also induced binding to this In a of the binding of and to 2 and could be but not to with EGF not the binding of RARs not In the chromatin of the human Has2 promoter a response to with all-trans-RA not RARs but also the of EGF In EGF a of binding to region The with in vitro binding of STAT protein to in promoter region of to the Has2 to the of the human Has2 gene via located in the proximal promoter the retinoid response of the gene turned to be associated with the of the Has2 by ChIP assays proteins that are involved in signal of retinoid receptors These were the receptor RXR, the histone the mediator protein Pol II, pPol II, and also histone 4 as a of promoter region 5 a in the first 1, and region 4 as a for the of the used were HaCaT The region to the proteins a binding of histone associated to 2 and and the binding all-trans-RA In binding found to four of the of which region the to and but associated in a with promoter and Pol II a but binding to 2 and and a with and region associated much with Pol II region and a of with all-trans-RA. important the with pPol II, which for region the with all-trans-RA with Pol region a induced binding of pPol II at the and a at Interestingly, 2 and a binding of Pol II, pPol II, at over the of the which monitored by histone 4 found to be high for the but in the of region Taken together, the of nuclear proteins with the four of the Has2 promoter complex the of nuclear proteins. the of retinoid signaling J. C. 2004; PubMed Scopus Google the of to and as for the of all-trans-RA and a of pPol II but to the all-trans-RA region 2 as the most promoter region the of all-trans-RA Has2 gene The and of epidermis a between keratinocyte proliferation and differentiation, in such as and turned that synthesis and concentration of hyaluronan are associated with these and epidermal keratinocyte Moreover, of cells is by hyaluronan and the of which be a of and the of which in Tammi M.I. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). is important to the of hyaluronan synthesis in it is that growth factors and hyaluronan synthesis and of their signaling are the hyaluronan synthesis at the chromatin J. J. M. T. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). with the of that the concentration of hyaluronan in cells and is correlated with Has gene in with the of Has2 (4Pienimäki J.P. Rilla K. Fulop C. Sironen R.K. Karvinen S. Pasonen S. Lammi M.J. Tammi R. Hascall V.C. Tammi M.I. J. Biol. Chem. 2001; 276: 20428-20435Abstract Full Text Full Text PDF PubMed Scopus (166) Google A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the between and of REK cells in organotypic and the of In the HaCaT human keratinocyte cell demonstrated that the human Has2 gene is also a primary EGF and all-trans-RA responding are in the of skin a primary RA responding in human keratinocytes J. PubMed Scopus Google Scholar). the of Has2 2 h with all-trans-RA is a response with that of the gene A. PubMed Scopus Google which is the most primary RA gene that the Has2 gene could be used as a for the of in Rev. 2001; PubMed Scopus Google Scholar). the to and in HaCaT cells in REK and of the that the human but not the rat is in the first 10 kb of the human Has2 promoter for and in vitro gel shift identified an with the of bp in of the bp to J. J. M. T. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google in the proximal region of the Has2 In the in the in vitro in vitro confirmed by ChIP of Has2 promoter In HaCaT cells region 1, to to the TSS, associated with a with EGF, the promoter region promoter region the region of the Has2 promoter that associated with activated EGF that the is and the mediator of the Has2 the phosphorylation of STAT proteins by activated EGFRs to be the most EGF signaling it not be that also signaling such as and PubMed Scopus Google Scholar). also to the is by a binding J. J. M. T. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google it be that STAT and proteins this in silico for RAREs in four not binding in is of the that the are of the in vitro gel shift are and protein were in reporter gene assays the RARE by RAREs and 2 to an all-trans-RA-dependent reporter gene that with that of the RARE. We found of the receptor to Has2 promoter to that not RARE confirmed by using a not of retinoid signaling J. C. 2004; PubMed Scopus Google Scholar) RARs are in complex with it is important that promoter 2 and with RXR, which these two as sites of retinoid signaling the Has2 The direct of the with proteins of in retinoid signaling demonstrated that the RARE containing region 2 a with protein and pPol II, activated Pol Moreover, at promoter region 2 could a of chromatin be Taken together, this evidence that promoter region to of the human Has2 is the region for the retinoid response of the with the of the RARE of nuclear receptor binding and within this promoter located bp in of the bp to J. J. M. T. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google the RARE not to be and not stringent gel shift in vitro binding of retinoid its functionality in reporter gene assays it that the retinoid response of the human Has2 gene is via a protein complex containing CBP, and activated Pol II associated with this RARE of nuclear receptor such as the J. C. J. J. C. Pharmacol. 2002; PubMed Scopus Google Scholar) or the C. A. PubMed Scopus Google Scholar) In REK with the kinase not in the of Has2 but in HaCaT cells it a of the basal expression of the that that were by HaCaT cells or of the basal of the Has2 which by the that the basal activity of the rat Has2 gene is that of the human Has2 a finding in with the between human and rat transcription factor binding sites within the first 10 kb of the Has2 The basal activity of the human Has2 gene could be the for its by EGF and all-trans-RA. Taken together, the rat and the human Has2 gene are by EGF and to in the of their regulation. The of the all-trans-RA-dependent of of the Has2 promoter with nuclear proteins that region 2 not the that to We found that all-trans-RA induced the recruitment of and to their binding sites in region this region not RARE In the ChIP that STAT associated also in a with and which not is a specific to retinoid that RARs to be the of these the in the ChIP is not proteins to but also proteins to it is to that one large protein complex is the Has2 protein complex Pol II or pPol II as its to transcription such as and RAR, and proteins. Moreover, this complex also enzymes with chromatin such as and The promoter this protein complex and be at are as transcription factor In this RARs proteins in the complex the of or the protein could be such a between RARs and STAT it with of Sci. S. A. 1997; PubMed Scopus Google Scholar). is that are to this of promoter In found that the rat and human Has2 are primary EGF and RA responding but that are in the of their regulation. The human Has2 gene contains a in its proximal promoter and a RARE kb upstream of the the retinoid response of the
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