Fos-related Antigen (Fra-1), junB, and junD Activate Human Involucrin Promoter Transcription by Binding to Proximal and Distal AP1 Sites to Mediate Phorbol Ester Effects on Promoter Activity

Abstract

Human involucrin (hINV) is a cornified envelope precursor that is specifically expressed in the suprabasal epidermal layers. We previously demonstrated that 2500 base pairs of the hINV gene upstream regulatory region confers differentiation appropriate regulation in transgenic mice. An analysis of the hINV gene sequence upstream of the transcription start site reveals five potential AP1 binding sites (AP1-1 to 5). Using reporter gene constructs in human keratinocytes, we show that the most distal (AP1-5) and most proximal (AP1-1) AP1 sites are essential for high level transcriptional activity. Simultaneous mutation of these sites reduces transcription by 80%. Gel supershift experiments indicate the interaction of these sites with Fra-1, junB, and junD. Involucrin mRNA levels increase 10-fold and promoter activity 5-11-fold when differentiation is induced by phorbol ester. Functional studies implicate AP1-1 and AP1-5 in mediating the phorbol ester-dependent increase in promoter activity. No involucrin promoter activity or involucrin mRNA was detected in 3T3 fibroblasts. We conclude that (i) two AP1 sites in the hINV promoter are important elements required for keratinocyte-specific expression, (ii) these AP1-1 sites mediate the phorbol ester-dependent increase in promoter activity, and (iii) Fra-1, junB, and junD may be important regulators of hINV expression in epidermis.

Opposing activities of c-Fos and Fra-2 on AP-1 regulated transcriptional activity in mouse keratinocytes induced to differentiate by calcium and phorbol esters

“…In contrast, the induction of the pro®laggrin promoter construct in TPA-treated cells appeared to be entirely dependent upon an intact AP-1 site. It is interesting that the silencing e ects of the AP-1 sequences on the involucrin promoter in calcium-treated human keratinocytes described above (Lopez-Bayghen et al, 1996) were not apparent in TPA-treated human keratinocytes (Welter et al, 1995). It is likely that c-Fos is induced under these conditions, and that the silencing e ects of other AP-1 family members are masked by the presence of c-Fos.…”

“…AP-1 DNA binding sites have been shown to play an important role in cell-and tissue-speci®c expression of the loricrin (DiSepio et al, 1995), involucrin (Crish et al, 1993;Welter et al, 1995), keratin 5 (Casatorres et al, 1994) and keratin 1 (Rothnagel et al, 1993;Lu et al, 1994) genes in vitro and in vivo. Additional in vitro studies indicate that AP-1 proteins largely play a positive role in regulating the expression of keratinocyte genes that harbor AP-1 sites (Navarro et al, 1995;Yamada et al, 1994;Takahashi and Iizuka, 1993;DiSepio et al, 1995;Bernerd et al, 1993;Oshima et al, 1990), although in vivo studies will ultimately be required to provide a de®nitive demonstration of the role of AP-1 in regulating gene expression in the epidermis.…”

Structure and chromosomal assignment of the mouse fra-1 gene, and its exclusion as a candidate gene for oc (osteosclerosis)

“…However, unlike c-Fos, Fra-1 lacks a transactivation domain and the entire Fra-1 protein fused to the DNA-binding domain of Gal4 was shown to lack any transcriptional activation function (Suzuki et al, 1991;Wisdom and Verma, 1993;Bergers et al, 1995). Therefore, fra-1 can not only increase, but also repress total AP-1 activity depending on the status of the other Fos proteins in the cell (Suzuki et al, 1991;Groskopf and Linzer, 1994;Bergers et al, 1995;Welter et al, 1995;Yoshioka et al, 1995). The transcriptional regulation of fra-1 is unique among members of the fos gene family, since only fra-1 is subject to positive control by AP-1 activity (BruÈ sselbach et al, 1995;Bergers et al, 1995).…”

Keratinocyte proliferation, differentiation, and apoptosis—Differential mechanisms of regulation by curcumin, EGCG and apigenin

“…Keratinocyte differentiating agents, including calcium and phorbol ester, activate involucrin gene expression Efimova et al, 2002;Efimova et al, 2003) via a signaling cascade that includes the novel PKC isoforms (nPKC), Ras, MEKK1, and MEK3 (Agarwal et al, 1999;Welter et al, 1995;Efimova and Eckert, 2000;Efimova et al, 1998). Activation of this cascade leads to an increase in p38δ and a decrease in ERK1/2 activity leading to an increase in the levels of AP1, Sp1 and CAATT enhancer binding protein (C/EBP) expression, increased binding of these factors to the hINV promoter, and increased hINV gene expression (Banks et al, 1998;Welter et al, 1995;Crish et al, 2002). Evidence for this cascade is provided by studies using pharmacological agents, constitutively-active and dominant-negative kinases, and kinase assays (Efimova and Eckert, 2000;Efimova et al, 1998;Efimova et al, 2002;Efimova et al, 2003).…”