Regulation of the human involucrin gene promoter by co-activator proteins

Abstract

Stratified squamous epithelial cells undergo an orderly process of terminal differentiation that is characterized by specific molecular and morphological changes, including expression of the cornified envelope protein involucrin. Significant progress has been made in characterizing the upstream regulatory region of the involucrin gene. Binding sites for AP-1 (activator protein 1) and Sp1 transcription factors were shown to be important for involucrin promoter activity and tissue-specific expression. Defective terminal differentiation is often characterized by decreased or lack of involucrin expression. Recently, a dominant-negative construct of the transcriptional co-activator P/CAF [p300/CBP-associated factor, where CBP stands for CREB (cAMP-response-element-binding protein)-binding protein] was shown to inhibit involucrin expression in immortalized keratinocytes [Kawabata, Kawahara, Kanekura, Araya, Daitoku, Hata, Miura, Fukamizu, Kanzaki, Maruyama and Nakajima (2002) J. Biol. Chem. 277, 8099-8105]. Loss of expression or inactivation of other co-activators has also been demonstrated [Suganuma, Kawabata, Ohshima, and Ikeda (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 13073-13078]. In the present study, we re-expressed CBP and P/CAF in immortalized keratinocyte lines that had lost expression of these co-activator proteins. Re-expression of these proteins restored calcium- and RA (retinoic acid)-responsive involucrin expression in these cells. RA and calcium signalling induced exchange of CBP and P/CAF occupancy at the AP-1 sites of the involucrin promoter. CBP and P/CAF inductions of the involucrin expression were not dependent on MEK (mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase), p38, protein kinase C or CaM kinase (calcium/calmodulin-dependent kinase) signalling. Kinase-induced changes in involucrin promoter activity directly resulted from changes in AP-1 protein expression. We concluded that CBP and P/CAF are important regulators of involucrin expression in stratified squamous epithelial cells.

Figure 1. Re-expression of CBP or P/CAF restores the regulation of involucrin expression in co-activator-negative cell lines

(A) Expressions of CBP and P/CAF (PCAF) proteins in immortalized lines and the normal epithelial strain NHEK were determined by Western-blot analysis as described in the Materials and methods section. Representative blots are shown. (B) Expression of CBP and P/CAF proteins was determined by Western-blot analysis in three stable clones transfected with CBP expression vector (CBP-1, CBP-2, CBP-3) or P/CAF expression plasmid (P/CAF-1, P/CAF-2, P/CAF-3) as described in the Materials and methods section. Lack of expression in neomycin-resistant control clones is shown. Representative blots are shown. (C) Regulation of involucrin expression by 24 h exposure to 2 mM calcium (+Ca) or 1 μM RA (+RA) is restored in CBP-3 and P/CAF-3 stable clones. Control cultures were treated with 0.9% saline (−Ca) or 0.1% ethanol (−RA) vehicle for the same time period. Lack of effect of calcium and RA on a neomycin-resistant control clone is shown. Involucrin expression was similar in 90% (pre) confluent and 2 days post-confluent cultures. Representative blots are shown. These experiments were repeated three times with similar results.

Figure 2. CBP and P/CAF restore calcium- and RA-dependent regulation of the involucrin promoter

(A) Triplicate cultures of SCC12 cells were transiently transfected with the involucrin promoter construct (pINV-luc) or the same plasmid containing mutations in the −125 (pINVmAP1.1) or −2122 (pINVmAP1.5) AP-1 site. Point mutations were also constructed in the −2113 Sp1 site (pINVmSp1) or all the three sites (B, D; pINVmAP1Sp1). The reporter plasmid was co-transfected with CBP or P/CAF expression vectors as described in the Materials and methods section. Cultures were treated with vehicle or 1 μM RA [RA, CBPR (CBP+RA), PCFR (P/CAF+RA)] for 24 h before harvesting for reporter gene analysis. (C) Cultures were transfected as described above and treated with vehicle or 2 mM CaCl₂ [Ca, CBPC (CBP+calcium), PCFC (P/CAF+calcium)] for 24 h before harvesting for reporter gene analysis. Relative light units (RLU) were normalized to the activity of a co-transfected β-galactosidase vector. These experiments were performed three times with similar results. Error bars represent S.E.M.

Figure 3. RA or calcium treatment induces differential AP-1 site occupancy by CBP and P/CAF

SCC12 clones expressing both CBP and P/CAF were treated with vehicle, 1 μM RA or 2 mM CaCl₂ for 30 min to 4 h before being subjected to ChIP using anti-CBP (IP CBP) or anti-P/CAF (IP PCAF) antibodies as described in the Materials and methods section. Relative occupancy of the −125 (PCR AP1-1) or −2122 (PCR AP1-5) AP-1 sites was determined by PCR amplification of immunoprecipitated genomic DNA using primers flanking these sites. Relative amounts of input genomic DNA in each sample was determined before immunoprecipitation using β-actin PCR primers. These experiments were performed three times with similar results. Representative blots are shown.

Figure 4. The ability of CBP and P/CAF to induce involucrin promoter activity is not dependent on calcium or MAPKs

Triplicate cultures of cells at 50% confluence were transiently transfected with the involucrin promoter construct along with CBP, P/CAF or blank expression vectors as described in the Materials and methods section. After a 16 h recovery period, cultures were treated with vehicle, 1 μM PKC inhibitor Go6976, 10 μM CaM kinase inhibitor KN62, 10 μM MEK inhibitor PD98059 or 5 μM p38 inhibitor SB203580 for 24 h. Luciferase activity was measured in RLU, followed by normalization for transfection efficiency. These experiments were performed three times with similar results. Error bars indicate S.E.M.

Figure 5. Fra-1, FosB and JunB proteins bind to the proximal and distal AP-1 sites of the involucrin promoter and immunoprecipitate with CBP

(A) Nuclear extract from SCC12 cells was incubated with radiolabelled oligonucleotide probes corresponding to the −125 (AP1-1) or −2122 (AP1-5) AP-1 sites of the involucrin promoter. Anti-AP-1 antibodies were used to identify specific components of the DNA–protein complexes. Unlabelled competitor or mutant oligonucleotides were included in some reactions to demonstrate the binding specificity. No extract was used as the negative control. The positions of the supershifted (SS) complexes and the free probe are shown. (B) CBP was immunoprecipitated from stable clones (IP CBP) as described in the Materials and methods section. Preimmune IgG was used as the negative control antibody (IP IgG). Immunoprecipitated proteins were subjected to Western-blot analysis with anti-Fra-1, FosB and JunB antibodies. Blots were incubated with anti-CBP antibody to determine the relative amounts of immunoprecipitated protein in each lane. These experiments were repeated three times with similar results. Representative gels and blots are shown.

Figure 6. Fra-1 inhibition and FosB induction mediate the effects of PKC and CaM kinase inhibition on the involucrin promoter

(A) SCC12F2 cells were treated with 1 μM Go6976, 10 μM KN62 or vehicle for 8–48 h. Whole cell lysates were subjected to Western-blot analysis using anti-Fra-1, anti-FosB and anti-JunB antibodies as described in the Materials and methods section. This experiment was repeated three times with similar results. Representative blots are shown. (B) Fra-1 and dominant-negative FosB induce involucrin promoter activity. Triplicate cultures of SCC12F2 cells were transiently transfected with the involucrin promoter construct (pINV-luc) along with Fra-1, dominant-negative FosB (dnFosB) or blank expression vectors as described in the Materials and methods section. Involucrin promoter constructs containing double-point mutations in one or both of the AP-1 sites (pINVmAP1.1, pINVmAP1.5, pINVmAP1.1/1.5) were also transfected. After 2 days, luciferase activity was measured in RLU, followed by normalization for transfection efficiency. These experiments were performed three times with similar results. Error bars represent S.E.M.