Requirement of an AP-1 Site in the Calcium Response Region of the Involucrin Promoter

Abstract

Involucrin is a major protein of the cornified envelope of keratinocytes that provides much of the structural integrity of the skin. The gene expression of this differentiation marker is induced by elevated extracellular calcium in cultured human keratinocytes. A 3.7-kilobase fragment of this gene contains the necessary elements to drive a luciferase reporter in a calcium-dependent manner. We have sequenced the upstream region of the involucrin promoter and localized a calcium response element that contains an activating protein-1 (AP-1) site (TGAGTCA). Mutation of this site abolished the promoter activation by calcium. Compared with cells grown in 0.03 mm calcium, the binding activity of factors within nuclear extracts from keratinocytes for this AP-1 site was enhanced 3-fold in cells grown in 1.2 mm calcium. Immunoelectrophoretic mobility shift (supershift) assays identified JunD, Fra1, and Fra2 as the major factors that bind to the AP-1 element. Western analysis of the proteins in the nuclear extracts showed that the levels of c-Jun, JunB, JunD, FosB, and Fra2 increased and the levels of c-Fos and Fra1 decreased slightly with calcium treatment. The effect of calcium on the involucrin promoter was enhanced synergistically by phorbol 12-myristate 13-acetate (PMA) in a protein kinase-dependent manner. In conclusion, calcium-regulated involucrin gene expression is mediated at least in part by AP-1 transcription factors. Involucrin is a major protein of the cornified envelope of keratinocytes that provides much of the structural integrity of the skin. The gene expression of this differentiation marker is induced by elevated extracellular calcium in cultured human keratinocytes. A 3.7-kilobase fragment of this gene contains the necessary elements to drive a luciferase reporter in a calcium-dependent manner. We have sequenced the upstream region of the involucrin promoter and localized a calcium response element that contains an activating protein-1 (AP-1) site (TGAGTCA). Mutation of this site abolished the promoter activation by calcium. Compared with cells grown in 0.03 mm calcium, the binding activity of factors within nuclear extracts from keratinocytes for this AP-1 site was enhanced 3-fold in cells grown in 1.2 mm calcium. Immunoelectrophoretic mobility shift (supershift) assays identified JunD, Fra1, and Fra2 as the major factors that bind to the AP-1 element. Western analysis of the proteins in the nuclear extracts showed that the levels of c-Jun, JunB, JunD, FosB, and Fra2 increased and the levels of c-Fos and Fra1 decreased slightly with calcium treatment. The effect of calcium on the involucrin promoter was enhanced synergistically by phorbol 12-myristate 13-acetate (PMA) in a protein kinase-dependent manner. In conclusion, calcium-regulated involucrin gene expression is mediated at least in part by AP-1 transcription factors. involucrin activating protein-1 base pair(s) kilobase(s) polyacrylamide gel electrophoresis polymerase chain reaction phorbol 12-myristate 13-acetate keratinocyte growth medium phosphate-buffered saline electrophoretic mobility shift assay protein kinase C The skin is a dynamic organ composed of a dermal and an epidermal layer. The epidermis can be divided morphologically into four layers (1Bikle D.D. Pillai S. Endocr. Rev. 1993; 14: 3-19PubMed Google Scholar). The basal layer contains the actively proliferating cells. As the keratinocytes migrate from the basal layer outward, numerous differentiation markers, such as involucrin (INV)1 (2Eckert R.L. Green H. Cell. 1986; 46: 583-589Abstract Full Text PDF PubMed Scopus (321) Google Scholar), transglutaminase (3Thacher S.M. Rice R.H. Cell. 1985; 40: 685-695Abstract Full Text PDF PubMed Scopus (401) Google Scholar), loricrin (4Steven A.C. Bisher M.E. Roop D.R. Steinert P.M. J. Struct. Biol. 1990; 104: 150-162Crossref PubMed Scopus (170) Google Scholar), keratins K1 and K10 (5Li L. Tucker R.W. Hennings H. Yuspa S.H. Cell Growth Differ. 1995; 6: 1171-1184PubMed Google Scholar), and filaggrin (6Fleckman P. Dale B.A. Holbrook K.A. J. Invest. Dermatol. 1985; 85: 507-512Abstract Full Text PDF PubMed Scopus (105) Google Scholar), become apparent in the spinous and granular layers. The fully differentiated cells then form the cornified layer of the stratum corneum. In the outer layers of the epidermis, the cornified envelope is formed beneath the plasma membrane and is the product of extensive cross-linking of proteins such as INV, filaggrin, loricrin, small proline-rich proteins (7Robinson N.A. Lapic S. Welter J.F. Eckert R.L. J. Biol. Chem. 1997; 272: 12035-12046Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 8Kartasova T. van Muijen G.N. van Pelt-Heerschap H. van de Putte P. Mol. Cell. Biol. 1988; 8: 2204-2210Crossref PubMed Scopus (68) Google Scholar), elafin (9Alkemade J.A. Molhuizen H.O. Ponec M. Kempenaar J.A. Zeeuwen P.L. de Jongh G.J. van Vlijmen-Willems I.M. van Erp P.E. van de Kerkhof P.C. Schalkwijk J. J. Cell Sci. 1994; 107: 2335-2342Crossref PubMed Google Scholar), periplakin (10Ruhrberg C. Hajibagheri M.A. Parry D.A. Watt F.M. J. Cell Biol. 1997; 139: 1835-1849Crossref PubMed Scopus (178) Google Scholar), envoplakin (11Ruhrberg C. Hajibagheri M.A. Simon M. Dooley T.P. Watt F.M. J. Cell Biol. 1996; 134: 715-729Crossref PubMed Scopus (152) Google Scholar), and cystatin A (12Takahashi H. Asano K. Kinouchi M. Ishida-Yamamoto A. Wuepper K.D. Iizuka H. J. Biol. Chem. 1998; 273: 17375-17380Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) by transglutaminase and sulfhydryl oxidase. INV is one of the major proteins that is cross-linked by transglutaminase to form the cornified envelope. A 5.8-kilobase (kb) genomic fragment of the INV gene (ATCC 61047) contains two exons, of 43 and 2107 bp, respectively, an intron of 1188 bp, and 2461 bp of 5′-upstream DNA (2Eckert R.L. Green H. Cell. 1986; 46: 583-589Abstract Full Text PDF PubMed Scopus (321) Google Scholar). A 3.7-kb fragment of this genomic clone, which does not contain the coding region in the second exon, was subcloned into a β-galactosidase reporter vector, introduced into transgenic mice, and shown to be expressed only in the epidermis, indicating that this fragment contains the necessary signals to direct tissue-specific expression (13Carroll J.M. Albers K.M. Garlick J.A. Harrington R. Taichman L.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10270-10274Crossref PubMed Scopus (132) Google Scholar). Coincident with the higher expressions of both the INV and transglutaminase genes is an increase in the intracellular calcium level (14Menon G.K. Elias P.M. Arch. Dermatol. 1991; 127: 57-63Crossref PubMed Scopus (170) Google Scholar). Extracellular calcium raises the intracellular calcium (15Pillai S. Menon G.K. Bikle D.D. Elias P.M. J. Cell. Physiol. 1993; 154: 101-112Crossref PubMed Scopus (27) Google Scholar) and stimulates keratinocyte differentiation (16Pillai S. Bikle D.D. Mancianti M.L. Cline P. Hincenbergs M. J. Cell. Physiol. 1990; 143: 294-302Crossref PubMed Scopus (210) Google Scholar). The mRNA levels of both INV and transglutaminase are induced by elevated calcium in the culturing medium (17Su M.J. Bikle D.D. Mancianti M.L. Pillai S. J. Biol. Chem. 1994; 269: 14723-14729Abstract Full Text PDF PubMed Google Scholar, 18Ng D.C. Su M.-J. Kim R. Bikle D.D. Front. Biosci. (online). 1996; 1: a16-a24Crossref PubMed Google Scholar). The increase in mRNA level is due at least in part to an increase in transcription; therefore, calcium is important for the transcriptional regulation of differentiation markers such as INV. In this report, we have characterized the INV gene for the presence of calcium-dependent elements. We found that an AP-1 site in the upstream region of the INV gene is essential for the calcium response. The vector pGL3-basic (Promega, Madison, WI) contains the luciferase reporter gene but is not linked to any promoter. The reporter in pGL3-promoter (Promega) is regulated by the SV40 promoter, and the reporter in pGL3-control (Promega) is regulated by the SV40 enhancer and promoter. DNA used for transfection and DNA sequence determination was propagated in Escherichia coliJM109 and was prepared to the by the at the of was in to the cells. of phorbol 12-myristate 13-acetate prepared in was from and keratinocyte growth medium was from and phosphate-buffered saline was from from was by a 3.7-kb fragment that to bp of the INV gene from the (13Carroll J.M. Albers K.M. Garlick J.A. Harrington R. Taichman L.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10270-10274Crossref PubMed Scopus (132) Google Scholar) into the and of the pGL3-basic The of the and the INV promoter on the of the to the transcription site of the INV promoter. The 3.7-kb INV promoter was with and The of the and the of the vector DNA polymerase and to The of INV was to the of the of the and from the and from the by with and The then with polymerase necessary and to form the The site of was sequenced to sequence and by and The of site was by DNA The was by the intron of and The vector fragment was then with the of an by the and site was introduced to of this The to bp of the INV promoter. and by the to bp and to bp, respectively, from and The site is at bp and the are at and was by the to bp DNA fragment from the the was by of the region and bp by polymerase chain reaction The product was subcloned into The sequence of the region was by The and prepared by The upstream used to the of the of the INV and and respectively, as upstream The AP-1 site is and the base of is in The used was to with to bp of the INV gene from the site A DNA was in of at of a was to that the reaction mm mm mm mm of of of and of DNA polymerase in a The was for the sequence for for and for the of the the at for with and subcloned into the and of The DNA sequence of the region of was by The for transfection of human keratinocytes was from D.C. Su M.-J. Kim R. Bikle D.D. Front. Biosci. (online). 1996; 1: a16-a24Crossref PubMed Google Scholar, M. J. Invest. Dermatol. 1991; Full Text PDF PubMed Google Scholar). second human keratinocytes cultured from S. Bikle D.D. Hincenbergs M. Elias P.M. J. Cell. Physiol. 1988; 134: PubMed Scopus Google Scholar) and to a of cells in with 0.03 mm calcium. cells then with a of of an luciferase of (13Carroll J.M. Albers K.M. Garlick J.A. Harrington R. Taichman L.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10270-10274Crossref PubMed Scopus (132) Google Scholar), and of in of with 0.03 mm calcium for at in a The cells then with of in for with and in of with 0.03 mm calcium for cells the with calcium was in with two with and in reporter (Promega) the of with luciferase (Promega) and β-galactosidase The luciferase with the β-galactosidase and to the activity of cells with the luciferase which grown in with 0.03 mm calcium. The sequence of the bp and bp of the was in both an by the at the of The activating protein-1 (AP-1) and AP-1 and and and The AP-1 site of is and the of the AP-1 AP-1 with of in the presence of of kinase (Promega) for at activity of the DNA was analysis of this AP-1 element and and was by element into the site of the pGL3-basic vector the to region of the INV promoter. of the AP-1 sequence by an extracts prepared from cells cultured in with 0.03 1.2 mm calcium for a by and S.M. F.M. R. D.D. J.A. K. in and Scholar). in to by the and to the of was to a with of the of the to be mm mm mm mm of an with a sequence of and of AP-1 The at for and in gel electrophoresis in mm mm mm The was then by the gel to The in was by a The was from the in Welter J.F. J.F. C. Eckert R.L. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). of c-Fos Fra1 and Fra2 to of nuclear from keratinocytes grown in medium 1.2 mm calcium and at for then with AP-1 as of nuclear was in mm and mm at for in gel electrophoresis and then a The on The membrane was in with and for The membrane was then with and to c-Jun, JunB, JunD, FosB, Fra1, and Fra2 for at of the and to the with a of in for at The membrane was then four in with for The membrane was then with for at and four in with for The membrane was then with and an was on A 3.7-kb fragment of the INV gene was subcloned in of a luciferase reporter in the pGL3-basic The reporter activity of the vector was not by calcium the reporter activity of cells with the was enhanced to an of in a with calcium The of used for reporter activity was in the of the assay and the of the 3.7-kb promoter within of a vector reporter activity is on a SV40 promoter not The INV mRNA level of the keratinocytes was calcium treatment. luciferase activity of the reporter was at this which was the that a and increase in promoter activity was at mm calcium and at 1.2 and mm calcium higher of calcium increased promoter with 1.2 the calcium of 0.03 and 1.2 mm calcium used in to the of calcium on the of the INV A region was identified by of the INV promoter. of to bp to bp enhanced the and basal 3-fold the by calcium was not to and the intron to to contain one but calcium-dependent element. The of the INV promoter was characterized by and the showed calcium-dependent activation of the luciferase gene of the four that not have the region and basal and promoter this region to contain both calcium-dependent and enhancer elements. the enhancer activity of the of two of the region and bp not the reporter activity of cells grown in 0.03 1.2 this region does not contain any major basal calcium-dependent element. of to bp enhanced the basal reporter activity indicating the presence of a and the effect of calcium on the reporter was from to the in calcium-dependent activation be due to in the of calcium to such basal the presence of an enhancer element in this region that is important for the calcium-dependent activation of the INV the calcium both calcium response to and to and the promoter was that the calcium response region and bp be calcium in the medium induced reporter of this to DNA sequence of the upstream region is shown in A. and by both the basal and but the of on calcium with the at and bp a and calcium the calcium response was with of the The of the of and the of the with DNA We in the activity of the reporter The in activity is a of the in basal activity with the 3.7-kb was used as a in the of the region and bp an AP-1 site at to the of the AP-1 site in the calcium the AP-1 site introduced into in both the and at the site of the vector, to the INV transcription The AP-1 site of the INV gene to be in both in that the calcium of the and to the level of the of the to bp not the reporter but the to a C at bp in the abolished calcium to the level of the the AP-1 site at to bp is in keratinocytes and is essential for the promoter the binding of this AP-1 the INV AP-1 site used as in extracts of the cells grown in 0.03 and 1.2 mm calcium of nuclear extracts to the AP-1 in two The of was higher in the gel nuclear extracts from cells grown in 1.2 mm calcium was in the nuclear extracts from cells grown in 0.03 mm calcium, that the AP-1 binding factors elevated in cells grown in 1.2 mm calcium. The to be a of binding of nuclear factors to the AP-1 of abolished binding to the The was in that by the was of a of the AP-1 site in of AP-1 with a in a of binding to the We then the two on involucrin promoter The AP-1 element as as the and of this element subcloned into a reporter that the bp of the INV promoter. the AP-1 sequence enhanced the calcium both the and this activity to the INV promoter in a calcium-dependent the to bind to nuclear the AP-1 site the of phorbol we the and calcium on the INV gene was into keratinocytes grown in 0.03 at and 1.2 mm calcium was to the cells. calcium response was by and the promoter activity elevated for at least not activation of the INV gene levels by and elevated for at least both increased the INV promoter of calcium and enhanced reporter and cells with both the promoter activity was induced synergistically to The of calcium and abolished by of the cells with of a protein kinase C is to be in both the calcium and activation of the INV promoter. base of the AP-1 site in and in of promoter activity not therefore, we the of the AP-1 site in We the of the (AP-1) of the INV AP-1 element to on the SV40 promoter, a INV promoter that does not contain the AP-1 and a INV promoter that contains the AP-1 enhanced the SV40 promoter and the INV promoter but the INV promoter The calcium effect was the INV AP-1 element was that both the and the AP-1 are necessary for calcium regulation of the INV The regulation by calcium of the INV promoter is from that by of the AP-1 site to the SV40 promoter of pGL3-basic enhanced the promoter activity by to that of calcium. this AP-1 element increased the activation of the INV promoter activity by In the presence of the INV promoter, increased the promoter activity by the calcium effect on the effect on the promoter activity was the of the AP-1 site of the region and in the INV promoter the of the promoter both the and the AP-1 to be for calcium, promoter activity with only one AP-1 and to have on the levels of the AP-1 The factors in the nuclear extracts that bind to the AP-1 element identified to c-Jun, JunB, JunD, FosB, Fra1, and for JunD, Fra1, and Fra2 indicating that proteins are in the by extracts of cells by to the of the in the nuclear the of the proteins by have As a only Fra1 is in this nuclear Western of the nuclear extracts of keratinocytes showed the presence of c-Jun, JunB, JunD, FosB, Fra1, and Fra2 in keratinocytes in Western and identified in the keratinocytes. but one of the by and can be by of the by The is the in the Western which was only with the higher be of the AP-1 In a and for in an increase in levels of the and of c-Jun, and of JunB, a of JunD, a of FosB, and a of The levels of the of c-Fos and the of Fra1 slightly by calcium. The and of c-Jun, the and of and a of c-Fos The Western shown in are of from keratinocyte The levels of AP-1 proteins as by are We in the basal levels of and in the nuclear from one of keratinocyte in which the by calcium and was is the of the but in the of the keratinocyte have to this and to have on the AP-1 The of of the decreased with of treatment. The only is that the is that of the cells. The for the are The of increased with but the and decreased with treatment. The of JunD, the of the of Fra1, and the of Fra2 increased with the was with and calcium in of the and of and the of In the of JunD, FosB, and the calcium not the levels of proteins as with cells that only the treatment. In JunB, JunD, and Fra2 to be increased by both calcium and the to have on the levels of FosB, and extracts from cells with calcium for by and Western was nuclear extracts from cells grown in 1.2 mm calcium for with that from cells grown in 1.2 mm calcium for not The expression of the INV mRNA in human keratinocytes is induced by calcium in a and (17Su M.J. Bikle D.D. Mancianti M.L. Pillai S. J. Biol. Chem. 1994; 269: 14723-14729Abstract Full Text PDF PubMed Google Scholar, M. R. J. Invest. Dermatol. 1994; Full Text PDF PubMed Scopus Google Scholar, Bikle D.D. J. Invest. Dermatol. 1996; Full Text PDF PubMed Scopus Google Scholar). from nuclear assays that calcium INV mRNA The of the reporter is that for the The in the of luciferase and the INV mRNA have to such The of the INV promoter in the reporter that of the have to this to that of by calcium in reporter activity the 3.7-kb genomic fragment of the INV gene was used to drive the luciferase promoter in keratinocytes. the 3.7-kb fragment contains the necessary transcription elements to calcium of the INV promoter. a of and we have localized a calcium response element within a region of the INV region contains a DNA sequence that is important for the tissue-specific expression of the INV gene in keratinocytes. sequence of this region slightly from a sequence of the human INV gene A. A. P. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). for one sequence with that from a J.F. Eckert R.L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). with the elements in a and bp of the transcription to the calcium of the INV promoter. A identified an and bp from the transcription site that be for this effect J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). the INV element not the calcium-dependent promoter activity in the and not The of the site does not to have direct with the calcium response of of the region the presence of enhancer elements. and a in both the basal and reporter of the fragment to bp in the of calcium we that a calcium-dependent element is within this of the sequence of the region an AP-1 site at to We have found that this AP-1 site is important for calcium of the INV this AP-1 site in a and to calcium In to the INV the AP-1 element to be important for calcium activation of the K1 gene J.A. D.A. M.A. Roop D.R. J. Invest. Dermatol. 1993; Full Text PDF PubMed Google Scholar, Yuspa S.H. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar), and AP-1 elements are in loricrin, and keratins R.L. J.F. Welter J.F. J. Invest. Dermatol. 1997; Full Text PDF PubMed Scopus Google Scholar). The AP-1 proteins in keratinocytes to the transcription of differentiation markers, and regulation of the levels and of factors be a in the regulation of differentiation in the keratinocytes. The AP-1 site we identified as a calcium-dependent element was as a site J.F. J.F. C. Eckert R.L. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), but the of calcium and on the AP-1 site to The of by calcium and both to the activation was The of as with that of calcium that of the calcium be mediated by activation of the of calcium and by a the by the was of the calcium effect both calcium and to have on the INV promoter to both the AP-1 site in the bp of the INV promoter and the AP-1 site at to of the AP-1 site the AP-1 site in the in of calcium In the AP-1 site was to the both AP-1 the by on the INV promoter was the the sequence of the AP-1 element a in the calcium of the INV We identified JunD, Fra1, and Fra2 by as proteins that bind to the AP-1 element in cells with 1.2 mm calcium. J.F. J.F. C. Eckert R.L. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar) identified JunB, JunD, and Fra1 in cells. culturing we only to Fra1 as one of the proteins that to the AP-1 element the treatment. the AP-1 factors induced by calcium and are to the AP-1 element of the involucrin The apparent be due to the the two the identified the AP-1 factors that bind to the involucrin promoter AP-1 element the of the Western the protein levels of the AP-1 factors in the of binding to the involucrin promoter. we to to a in AP-1 factors binding to the AP-1 site calcium and calcium and to activity of the AP-1 proteins induced such as The of calcium and in the levels and activity of and We have that the of of the factors that bind to the AP-1 element higher in cells grown in elevated calcium. The of and proteins the layers of the human epidermis J.F. Eckert R.L. 1995; Google Scholar). In Fra1, and protein levels higher in the layers of the epidermis, the site of INV and increased calcium. we that the protein levels of the and Fra2 proteins increased with calcium in in factors be for the calcium of differentiation markers such as INV. In but not calcium, induced c-Fos and the increased levels of two proteins found in the layers of the epidermis be a of a such as that protein kinase C that is of In the cultured JunB, JunD, Fra1, and Fra2 elevated with calcium and c-Jun, JunB, JunD, and Fra1 induced by A. Yuspa S.H. 1996; Google Scholar, S. Yuspa S.H. 1997; PubMed Scopus Google Scholar). the for AP-1 proteins are in both the and human is induced by in but not human keratinocytes. Fra1 is elevated by both and calcium in keratinocytes but is only by in human keratinocytes. In only calcium can the Fra2 protein level in both calcium and can increase the Fra2 protein level in human keratinocytes. The of the is the growth factors in the culturing medium have the activity and expression of the AP-1 factors and to the the human and keratinocytes in calcium and have slightly on the AP-1 proteins in and human the that of the AP-1 protein are by calcium and calcium stimulates c-Fos expression in cells Mol. Cell. 1995; 107: PubMed Scopus Google Scholar), granular J. 1995; PubMed Scopus (27) Google Scholar), the M. C. Mol. Cell. 1996; PubMed Scopus (26) Google Scholar), and cells M. Mol. Cell. 1996; PubMed Scopus Google Scholar). are to be mediated by of the response element binding protein by protein and activation of the response element in the c-Fos promoter M. M.A. M.E. 1991; PubMed Scopus Google Scholar, M. M.E. 1990; Full Text PDF PubMed Scopus Google Scholar, H. H. 1995; PubMed Scopus Google Scholar, Mol. Cell. Biol. 1994; 14: PubMed Scopus Google Scholar). be for the in levels of the c-Fos AP-1 proteins in keratinocytes calcium treatment. INV is an essential protein in the epidermis, and calcium is for epidermal We have identified an upstream region that contains a of important for expression of the an AP-1 element to be essential for the of the INV gene by calcium. and elements are to be for expression of the differentiation markers in the epidermis We for of human J. and C. L. for and J. and L. of for sequence of the INV promoter and the β-galactosidase