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.The skin is a dynamic organ composed of a dermal and an epidermal layer. The epidermis can be divided morphologically into four layers (1). 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 (2), transglutaminase (3), loricrin (4), keratins K1 and K10 (5), and filaggrin (6), 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 (7,8), elafin (9), periplakin (10), envoplakin (11), and cystatin A (12) 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 5Ј-upstream DNA (2). A 3.7-kb fragment of this genomic clone, which does not contain the coding region in the second exon, was subcloned into a ␤-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 (13).Coincident with the higher expressions of both the INV and transglutaminase genes is an increase in the intracellular calcium...

The human involucrin gene contains spatially distinct regulatory elements that regulate expression during early versus late epidermal differentiation

“…All of the known AP1 factors are expressed in both human and mouse epidermis, each displaying a speci®c pattern of di erentiation-dependent expression (Rutberg et al, 1996;Briata et al, 1993;Fisher et al, 1991). Because the AP1-5 site has been speci®cally implicated as an important regulator of involucrin promoter activity in cultured cells (Takahashi et al, 1998;Takahashi and Iizuka, 1993;Banks et al, 1998Banks et al, , 1999E®mova and Eckert, 2000;Ng et al, 2000;E®mova et al, 1998), we chose to evaluate its role as a di erentiation regulator. Our results show that mutation of the AP1-5 site results in markedly reduced expression at all stages of di erentiation.…”

Calcium-dependent Involucrin Expression Is Inversely Regulated by Protein Kinase C (PKC)α and PKCδ

“…PKC and Calcium Regulate hINV Gene Expression-Previous studies (29,60,61) suggest that calcium regulates hINV gene expression at the mRNA and protein level and suggest that novel PKC isoforms mediate the phorbol ester-dependent increase in hINV gene expression. The PKC regulation is transmitted via a pathway that includes novel PKC, Ras, MEKK1, MEK3, and p38 MAPK (28,29).…”

Function and regulation of AP-1 subunits in skin physiology and pathology

“…High basal level expression of the pro®lag-grin gene in epidermal keratinocytes depends on c-Jun/ c-Fos heterodimers (Jang et al, 1996), whereas cooperative action of AP-1 and Sp1 is required for maximal expression of the human involucrin promoter (Lopez-Bayghen et al, 1996). JunD, Fra-1 and Fra-2 proteins, whose levels are a ected by changes in the concentration of extracellular calcium, preferentially bind to the calcium response element in this promoter (Ng et al, 2000). However, Fra-1, JunB and JunD appear to be responsible for the regulation of involucrin promoter activity by phorbol esters .…”