©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 (*)

Jean F. Welter (1)(§), James F. Crish (1)(¶), Chapla Agarwal (1), Richard L. Eckert (1) (2) (3) (4) (5)(**)

From the (1) Departments of Physiology and Biophysics, (2) Dermatology, (3) Reproductive Biology, (4) Biochemistry, and (5) Oncology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

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.


INTRODUCTION

The human epidermis is a keratinizing squamous epithelium consisting of several distinct layers (for review, see Ref. 1). The basal layer is located adjacent to the dermis and consists of relatively undifferentiated, proliferative cells. During the process of terminal differentiation the keratinocytes, responding to unknown stimuli, withdraw from the cell cycle, migrate from the basal to the superficial layers of the epidermis, and simultaneously undergo morphological and biochemical changes. The terminal cell consists of a network of cytokeratin filaments surrounded by an insoluble envelope of heavily cross-linked protein (2, 3) . The envelope components and transglutaminase, the enzyme responsible for assembly, must be expressed at the proper time and level during differentiation for the envelope to be formed properly (1, 4, 5, 6, 7, 8, 9) . One of these components expressed specifically in the suprabasal layers of the epidermis is involucrin. Involucrin is a highly reactive, soluble, transglutaminase substrate, which functions as a glutamyl donor during assembly of the cornified envelope (4, 6, 10, 11, 12, 13, 14, 15) .

Little is known about mechanisms responsible for tissue- and differentiation-specific regulation in most systems. In skin, however, evidence implicating the protein kinase C-AP1 pathways is rapidly accumulating (16, 17, 18) . AP1 was initially described as a DNA binding activity in HeLa cell extracts that recognized specific sites in the SV40 enhancer (19) . Specific DNA binding sites have since been identified in a wide variety of genes (20, 21) . The term AP1 describes a broad and fairly ubiquitous class of transcription factors that consist of either a homo- or heterodimer of two jun family proteins, or a heterodimer of one jun and one fos family protein (22) . Three members of the jun family, c-jun, junB (23) , and junD (24, 25) have been identified, while the fos family includes c-fos, fosB (26) , fosB2, Fra-1 (27, 28) , and Fra-2 (29, 30) . These proteins appear to be expressed differentially in various cell types, can form heterodimers with each other, and bind the same DNA consensus sequence. However, they respond differently to activating stimuli (31-34), and they appear to have differing effects on cell function (30, 35).

Several findings have lead to speculation about a possible regulatory role for AP1 proteins in the epidermal differentiation process. In most quiescent adult human tissues, levels of the AP1 component c-fos, for example, are extremely low, but are rapidly induced by mitogenic stimuli (36) . In contrast, fos is constitutively expressed at high levels in the non-proliferative differentiating suprabasal epidermal layers (16, 18) . In addition, AP1 has recently been implicated in the regulation of several keratinocyte genes (37, 38, 39) .

Transgenic studies from our laboratory suggest that a 2.5-kilobase fragment of the human involucrin gene (hINV)() upstream of the transcription start site contains all of the information required for tissue-specific and differentiation-appropriate targeting to the epidermis (40) . In the present study we (i) identify five AP1 sites within this upstream regulatory region and confirm that two of these sites are necessary for expression of the gene in keratinocytes, (ii) demonstrate that the promoter is inactive in fibroblasts, (iii) show that the AP1 transcription factors Fra-1, junB, and junD are major contributors to the protein complexes which bind at these two sites, and (iv) show that these sites are important mediators of the inductive response observed following treatment with phorbol esters.


MATERIALS AND METHODS

Chemicals and Reagents

Serum-free keratinocyte media (KSFM), trypsin, Hanks' balanced salt solution, gentamicin, and Lipofectin were obtained from Life Technologies, Inc. The plasmids pGL2-Basic, pGL2-Control, and pSV--galactosidase, the GL-2 polymerase chain reaction primer, AP1 and SP1 consensus sequence oligonucleotides, and the chemiluminescent luciferase assay systems were obtained from Promega. The T/A cloning vector, pCRII, and competent INV-F` bacteria were purchased from Invitrogen. The Galactolight -galactosidase chemiluminescent assay system was obtained from Tropix. Dispase was obtained from Boehringer Mannheim, [-P]ATP from DuPont NEN. Phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA) and dimethyl sulfoxide were from the Sigma. Chemiluminescence was measured using a Berthold luminometer and oligonucleotides were synthesized on an Applied Biosystems DNA synthesizer. Transcription factor binding sites in the hINV promoter were identified using the Signal Scan DNA sequence analysis program (41) .

Antibodies

Antibodies against transcription factors were purchased from Santa Cruz Biochemicals and were rabbit polyclonal antibodies raised against synthetic peptides. The broad spectrum anti-c-fos antibody (pan-fos) was raised against the highly conserved c-fos amino acids 128-152, and reacts with c-fos, fosB, Fra-1, and Fra-2. The broad spectrum anti-c-jun antibody (pan-jun) was raised against amino acids 247-263 of mouse c-jun, and cross-reacts with c-jun, junB, and junD. The remaining antibodies used in this study (c-fos, fosB, Fra-1, Fra-2, c-jun, junB, and junD) were raised against sequences specific to each protein and do not cross-react.

Plasmid Construction

The plasmids used in this study include pSP64I-3 H/Hc, which contains a HindIII/HincII fragment of the hINV gene including approximately 2.5-kilobase pairs of upstream regulatory sequence, the TATA box, and the hINV intron (12) , pUC1813 (42) , and pGL2-Basic, a promoterless luciferase reporter vector. To construct plasmids for the deletion series, a 2.4-kilobase pair HindIII/CelII fragment of the hINV gene including positions -7 to -2473 relative to the transcription start site was released from pSP64I-3 H/Hc. The ends of this fragment were made blunt with Klenow polymerase prior to cloning into SmaI-restricted pUC1813 to produce pUC1813I-3 HindIII/CelII, in which the hINV segment is flanked by BamHI sites. The BamHI fragment encoding the hINV sequence was then subcloned into BglII-restricted pGL2-Basic. The resulting clones were screened for the appropriate orientation of the inserted DNA fragment by restriction mapping. The plasmid containing the hINV insert in the appropriate orientation was named pINV-2473 based on the nucleotide at the most 5` end of the hINV segment, as determined by counting backwards from the start site of transcription of the hINV gene (12) . Maps of the hINV promoter-luciferase fusion constructs are shown in Fig. 1. The other plasmids in the series were produced by removing promoter segments using restriction enzymes, or by reconstructing gene segments using the polymerase chain reaction, and are also named according to the 5` most nucleotide from the hINV sequence.


Figure 1: Human involucrin promoter-luciferase reporter gene fusion constructs. In each map, the narrow line represents the hINV sequence and the dark box symbolizes the luciferase gene. The arrow indicates transcription of the luciferase product. The plasmids are named (pINV-2473, pINV-2216, etc.) based on the length of the segment, as measured backwards from the hINV transcription start site. The five AP1 sites present within the upstream sequence are numbered 1 to 5 and indicated by circles. The scale at the top is in base pairs.



pINV-793 was used as a template to produce three constructs, pINV-679, pINV-489, and pINV-298, by polymerase chain reaction. Involucrin sequence-specific oligonucleotides served as upstream primers and a pGL2-Basic-specific primer, GL-2 (Promega), was used as the downstream primer. The polymerase chain reaction products were cloned into pCRII for further amplification and finally subcloned into pGL2-Basic.

To create the plasmids containing mutated AP1-1 and/or AP1-5 sites (see below, and ), convenient restriction sites flanking the AP1 sites were identified, and the fragments containing the wild type AP1 sites were released by restriction digestion. Oligonucleotides identical to the wild type sequences between these restriction sites, except for the mutated bases, were synthesized and ligated into the parent constructs. Conservative thymidine to cytosine mutations at position 5 of the consensus binding sequence were chosen (43) .

It is important to note that the junction between the hINV gene sequences and the luciferase reporter gene sequence are identical in all constructs.

Tissue Culture

Primary human keratinocytes were cultured from human foreskin samples. Foreskin specimens were stored overnight at 4 °C in KSFM containing 5 µg/ml gentamicin. The specimens were rinsed for several minutes in Hanks' balanced salt solution containing 20 µg/ml gentamicin, cleaned of connective tissue, and incubated for 18 h at 4 °C in Hanks' balanced salt solution containing 10 mg/ml dispase to separate the epidermis from the dermis (44) . The sheet of epidermis was then dissociated with 0.25% trypsin in Hanks' balanced salt solution containing 1.0 mM EDTA for 5 min at 37 °C. The trypsin was neutralized with serum and the cells were plated in KSFM at 1-3 10 cells per 10-cm dish. The cells were grown at 37 °C in a 5% CO atmosphere, split at a 1:5 ratio when 70% confluent, and used for transfection at the third passage. Mouse embryonic fibroblast 3T3 cells were grown as described previously (45) .

Transfection

Cells were transfected in 60-mm diameter dishes when 60% confluent. Sixteen micrograms of Lipofectin reagent, 3.5 µg of test plasmid, 0.5 µg of the pSV--galactosidase (an SV40 promoter/enhancer -galactosidase reporter vector, as an internal standard) were mixed, added to cells in 3 ml of KSFM, and incubated for 5 h at 37 °C. A parallel set of dishes was co-transfected with 3.5 µg of pGL2-Control (luciferase driven by the SV40 promoter/enhancer, Promega) and 0.5 µg of pSV--galactosidase. At 5 h, 3 ml of KSFM was added and the incubation continued for an additional 19 h. The cells were then allowed to recover for 24 h in fresh KSFM. At this point, the cultures were washed once with KSFM and then treated for 24 h with KSFM containing 50 ng of TPA/ml (from 5 mg/ml stocks in MeSO) to induce differentiation (46) .

Luciferase Assay

The cells were washed twice with phosphate-buffered saline, dissolved in 250 µl of cell culture lysis reagent (Promega), and harvested by scraping. Luciferase assays were performed immediately using a Berthold luminometer and the Promega luciferase assay kit. Cell extract (20 µl) and 100 µl of luciferin mixture were reacted for 5 s and light output was monitored over the next 10 s. -Galactosidase assays were performed on duplicate aliquots using the Galactolight reagent system. All assays were performed in triplicate, and each experiment was repeated a minimum of three times.

Normalization of Luciferase Activity

Within each experiment, differences in transfection efficiency were normalized to the -galactosidase internal standard by dividing the involucrin luciferase signal by the pSV--galactosidase signal. However, use of the -galactosidase signal was not adequate to normalize between experiments using different cell lines. For this reason, we divided the normalized luciferase activity of each involucrin construct by the normalized pGL2-Control luciferase activity and expressed the results in dimensionless, arbitrary units.

Preparation of Nuclear Extracts

Passage 3 keratinocytes were grown in KSFM until 80% confluent and were then treated with KSFM supplemented with 50 ng of TPA/ml for 24 h. Nuclear extracts were prepared at approximately 0.5-2.0 mg of protein/ml according to the method of Dignam et al.(47) or as described by Schreiber et al.(48) .

Gel Mobility Shift Assay

Band shifts were conducted by incubating a 20-µl reaction containing 15% glycerol, 75 mM KCl, 0.375 mM dithiothreitol, 0.375 mM phenylmethylsulfonyl fluoride, 12.5 mM NaCl, 0.1 µg/µl poly(dI-dC), 2.5 µg of nuclear extract, and 0.3 ng of radiolabeled DNA for 5 min at room temperature. The samples were immediately electrophoresed at 250 V for 1.5 h on 5% nondenaturing acrylamide gels using a one-fourth TBE running buffer, dried, and autoradiographed. For competition studies, radioinert DNA competitor was added at a 20- or 200-fold molar excess.

Gel Mobility Supershift

The reaction mixtures were assembled as outlined above without the P-labeled oligomer and incubated with antibodies specific for jun or fos family members (1 µg of antibody per 2 µg of nuclear extract) for 2 h at 4 °C. The P-labeled DNA oligomer was then added, incubated for 5 min at room temperature and then immediately electrophoresed on a 5% nondenaturing acrylamide gel. In control reactions, the antibody was replaced with bovine serum albumin (1 µg of bovine serum albumin/2 µg of nuclear extract).


RESULTS

hINV Promoter Deletion Series

The constructs shown in Fig. 1were transfected into cultured human epidermal keratinocytes, and assayed for the ability to drive production of luciferase activity (Fig. 2). pINV-2473 and pINV-2216 produce high levels of luciferase activity. Elimination of 80 base pairs of pINV-2136 reduces the activity by 45% and luciferase activity fluctuates between 30 and 55% of maximum for pINV-1336, -1261, -1152, -1091, -986, -793, -679, -489, -298, -241, and -159. However, truncation to position -41, pINV-41, results in a drop in activity to 2.5% of the level observed for plasmids pINV-2473 or pINV-2216.


Figure 2: Transcriptional activity of the intact hINV promoter. Passage three keratinocytes were grown until 60% confluent, then cotransfected with each hINV-luciferase test plasmid and an internal control plasmid, pSV--galactosidase, as described under ``Materials and Methods.'' Twenty-four hours after transfection, the cells were treated for 24 h with fresh KSFM containing 50 ng of TPA/ml. Extracts were prepared and assayed for luciferase and -galactosidase activity. hINV-luciferase activity was normalized using -galactosidase as an internal standard. The normalized luciferase activity is expressed in arbitrary units as defined under ``Materials and Methods.'' The x axis measures the length of the hINV DNA segment in base pairs. The data points from left to right correspond to the constructs shown in Fig. 1.



Location of AP1 Sites in hINV Promoter

The upstream regulatory region of the hINV promoter contains four motifs having homology to the AP1 binding site, 5`-TGANT(C/A)NN-3`, where N is any nucleotide (20) and one site similar to the putative AP1 motif 5`-GAGAGGAA-3` (49) . Their positions in the hINV upstream segment are shown in Fig. 1and their sequence in . In the deletion series, the drop in activity observed between -2216/-2136 and -159/-41 in Fig. 2 (open circles) corresponds to the loss of AP1-5 and AP1-1, respectively. Removal of AP1-2, -3, or -4 does not appear to significantly influence the promoter activity.

hINV Promoter Point Mutations

To more specifically analyze the contribution of AP1-1 and AP1-5 to the overall activity of the hINV promoter, we generated hINV promoter constructs in which the AP1-1 and/or AP1-5 site were inactivated (i.e. rendered unable to bind AP1) by changing the T at position 5 in the consensus sequence to C () (43) . As shown in Fig. 3A, the mutation of AP1-1 reduces the promoter activity (solid symbols) for each construct tested.


Figure 3: AP1-1, AP1-5, and hINV promoter activity. In panel A, constructs pINV-2473, pINV-986, pINV-793, pINV-298, pINV-241, and pINV-159 (data points left to right) were modified to mutate the AP1-1 site by replacing sequence AP1-1 with AP1-1 m (see Table I). The x axis measures the length of the hINV DNA segment in base pairs and the y axis measures arbitrary units of luciferase activity. The open square connected to each curve by a dotted line indicates the pINV-41, the basal promoter, which lacks an AP1 site. It is included to indicate the activity of the minimal hINV transcription unit. In panel B, construct pINV-2473 was modified to eliminate AP1-1, AP1-5, or AP1-1 and AP1-5 by replacing these sites with AP1-1m, AP1-5m, or AP1-1m and AP1-5m, respectively. Constructs were tested for activity by transfection into normal human keratinocytes which were then treated exactly as outlined as described in the legend to Fig. 2.



Similarly, to evaluate the role of AP1-5, cells were transfected with construct pINV-2473 (see Fig. 1) containing an intact or mutated AP1-5 site. Activity of this construct was compared to the activity of a pINV-2473 AP1-1 site mutant and an AP1-1/AP1-5 dual mutant (Fig. 3B). In the experiment shown in Fig. 3B, elimination of AP1-1 or AP1-5 resulted in a 60 and 20% decrease in activity, respectively. Elimination of AP1 consistently resulted in a greater loss of promoter activity than elimination of AP1-5; however, the actual percentages varied among keratinocyte strains. Simultaneous mutation of AP1-1 and AP1-5 results in an 80% decline in activity, suggesting that the contribution of each site to overall activity is additive. Interaction of Nuclear Proteins with the AP1-1 and AP1-5 Sites-To characterize the interaction of nuclear proteins with the AP1-like consensus sites in the hINV regulatory region, we performed gel shift and oligonucleotide competition experiments. The sequence of AP1-1 and -5 are shown in , along with the sequence of other oligonucleotides used in the gel shift experiments. AP1c is a commercially available oligonucleotide. The AP1c consensus binding sequence is identical to the involucrin sequence, but the flanking sequences are different. AP1-1m, AP1-2m, and AP1-5m are mutants of the hINV AP1-1, AP1-2, and AP1-5 sites, respectively, in which the T at position 5 has been changed to C. The change of T to C at this position has been reported to result in a loss of AP1 binding (43) .

Fig. 4 shows a gel mobility shift experiment in which keratinocyte nuclear extract was incubated with P-AP1-1 in the presence or absence of excess radioinert AP1-1, AP1-1m, AP1c, SP1c, AP1-2, or AP1-2m competitor. The P-AP1-1 oligonucleotide shifts a major band (arrow) that is competed by oligonucleotides AP1-1, AP1c, and AP1-2, but not by AP1-1m, SP1c, or AP1-2m. Additional competition experiments with P-AP1-1 and increasing amounts of radioinert AP1 indicate half-maximal competition at 20-50-fold molar excess (not shown).


Figure 4: Interaction of nuclear proteins with the AP1-1 binding site. P-AP1-1 (0.3 ng) was incubated with nuclear extract in the presence of 0, 20-, or 200-fold molar excess of AP1-1, AP1-1m, AP1c, SP1c, AP1-2, or AP1-2m. The incubations were then electrophoresed on nondenaturing gels, dried, and autoradiographed with intensifying screens at -70 °C. The arrow indicates the major AP1-1 binding activity. N indicates a lane in which nuclear extract was absent. The free (unbound) oligonucleotide is not shown in this experiment.



A mobility shift assay in which P-AP1-5 was incubated with radioinert AP1-5, AP1-1, AP1-1m, AP1-2, AP1-2m, SP1c, and AP2c is shown in Fig. 5. The results show specific binding of a major band (arrow) that is competed by AP1 site-encoding oligonucleotides (i.e. AP1-5, AP1-1, AP1-2, and AP1c), but not by non-AP1-encoding sequences (i.e. SP1 and AP2c). Some competition was observed when AP1-1m was present at a 200-fold molar excess, but a 200-fold molar excess of AP1-2m did not compete. Identity of Nuclear Proteins Which Bind to AP1-1 and AP1-5-To identify the factors that interact with the AP1-1 site, nuclear extracts were incubated with P-AP1-1 in the presence or absence of antibodies specific for various jun and fos family members (Fig. 6A). Lane N shows the migration of free P-AP1-1 (arrowhead near gel front) in the absence of extract and the minus lane indicates the shifted protein-DNA complex observed in the absence of antibody (long arrow). The broadly reactive jun and fos antibodies (pan), as well as antibodies specific for fos family member Fra-1 (F1) and jun family members junB (B) and junD (D), produced supershifted bands (short arrows). No shift was observed in samples treated with antibodies specific for c-fos (c), fosB (B), Fra-2 (F2), or c-jun (c). An identical pattern of supershifts is observed when AP1-5 is used as the radioactive probe (Fig. 6B), suggesting that Fra-1, junB, and junD are factors that bind to both AP1-1 and AP1-5.


Figure 5: Interaction of nuclear proteins with the AP1-5 binding site. P-AP1-5 (0.3 ng) was incubated with nuclear extract in the presence of a 0, 20-, or 200-fold molar excess of AP1-5, AP1-1, AP1-1m, AP1-2, AP1-2m, AP1c, SP1c, or AP2c. The incubations were electrophoresed on nondenaturing gels, dried, and autoradiographed. The arrow indicates the major AP1-5 binding activity, the arrowhead indicates free P-AP1-5 and N indicates a lane in which the nuclear extract was omitted.




Figure 6: Identity of nuclear factors that interact with the hINV promoter AP1-1 and AP1-5 sites. Nuclear extracts were incubated with 0.3 ng of P-AP1-1 (A) or 0.3 ng of P-AP1-5 (B) in the absence (-) or presence of antibodies against pan-fos (pan), c-fos (c), fosB (B), Fra-1 (F1), Fra-2 (F2), pan-jun (pan), c-jun (c), junB (B), or junD (D). The complexes were then electrophoresed on a 5% nondenaturing acrylamide gel. The long arrows to the left in each panel indicate the complex formed in the absence of antibody. The short arrows to the right indicate the supershifted complexes. The arrowhead near the gel front indicates free P-AP1 oligomer. Nuclear extract was omitted in the lane marked N.



Phorbol Ester-dependent Induction of hINV Gene Expression

As shown above, the AP1-1 and AP1-5 sites are functionally important in maintaining optimal transcription in cultures grown in the presence of the differentiating agent, TPA. Fig. 7 shows that TPA treatment (+) increases the level of AP1 activity which binds to the hINV AP1-5 site (and AP1-1, not shown) compared to nuclear extracts prepared from untreated(-) cultures. The level of AP1 binding activity increased between 25 and 200-fold depending upon the experiment. As AP1 has been implicated in mediating the effects of phorbol esters in a variety of systems (20) , this suggested that these sites may mediate the TPA-dependent induction of hINV mRNA expression that has been previously reported in keratinocytes (50) . We therefore further investigated the role of AP1-1 and AP1-5 by testing the activity of full-length (pINV-2473) constructs in which one or both sites are inactivated by point mutation. As summarized in Fig. 8, mutation of the AP1-1, AP1-5, or both sites simultaneously reduces both basal and TPA-stimulated transcriptional activity. In the case of mutation of AP1-5, the overall activity is decreased, but the fold induction by TPA remains stable. Inactivation of AP1-1, in contrast, results in a decrease in the fold induction following TPA treatment. Simultaneous inactivation of AP1-1 and AP1-5 resulted in a >50% reduction in the TPA response compared to the intact construct.


Figure 7: Phorbol ester increases AP1 binding to AP1 DNA response elements. Nuclear extracts were prepared from cells grown for 24 h in the presence (+) or absence (-) of 50 ng/ml TPA. P-AP1-5 (0.3 ng) was incubated with each extract in the presence or absence of 0, 20-, and 200-fold molar excess of AP1-5 or AP1c. The extracts were then electrophoresed on nondenaturing gels, dried, and autoradiographed with intensifying screens at -70 °C. The bracket indicates migration of the major AP1 binding activity. Nuclear extract was omitted in the lanes marked N. The arrowhead indicates migration of the free, unbound oligonucleotide.




Figure 8: Mutation of AP1-1 sites reduces hINV promoter response to TPA. Normal human keratinocytes were transfected with pINV-2473 (the full-length hINV promoter, Intact), or with variants in which AP1-5 (AP1-5m), AP1-1 (AP1-1m), or AP1-1 and AP1-5 (AP1-1m/5m) are inactivated by point mutation. The cells were then treated in the presence (+TPA) or absence (-TPA) of 50 ng/ml TPA for 24 h, harvested, and assayed for luciferase activity. All activities are normalized by setting the activity of the intact promoter in the absence of TPA to 100. The fold increase, X, indicates the ratio of activity in the presence of TPA/activity in the absence of TPA for each construct. The numbers at the top of the figure indicate the distance in base pairs along the hINV promoter and the black box indicates the luciferase reporter gene. The positions of AP1-1, -2, -3, -4, and -5 are indicated by the five open circles.



Tissue Specificity of hINV Promoter Activity and Phorbol Ester Response

As shown in Fig. 9, TPA significantly increases the level of human involucrin mRNA present in keratinocytes. In contrast, no basal expression and no TPA responsive induction of involucrin mRNA are observed in NIH 3T3 cells. Promoter transfection studies in keratinocytes indicate a 7.5-11-fold increase in promoter activity following TPA treatment for the pINV-2473 and pINV-241 constructs. In parallel experiments, no promoter activity and no TPA response were observed in 3T3 cells (Fig. 9). These results suggest that the promoter activity is restricted to keratinocytes and requires the AP1-1 and AP1-5 sites.


Figure 9: Phorbol ester regulation of involucrin promoter activity and mRNA level. Normal human keratinocyte (NHK) and 3T3 mouse fibroblast (3T3) cultures were treated in the presence (+) or absence (-) of 50 ng/ml TPA for 24 h. Poly(A) RNA was prepared, electrophoresed, and transferred to Biodyne A membrane as described previously (62). The membranes were hybridized with a P-labeled cDNA probes encoding human (NHK cells) or mouse (3T3 cells) involucrin (INV). The loading was normalized by hybridizing with an actin cDNA probe (A). Transcriptional activity of the human involucrin promoter (constructs pINV-2473 and pINV-241) was monitored as described under ``Materials and Methods'' and in the legend to Fig. 2. The activity bars shown in the 3T3 assay indicate background activity (i.e. the promoter was completely inactive in this cell type). The SV40 promoter, transfected in parallel as a positive transfection control, was active in both cell types. The mouse involucrin cDNA was kindly provided by Drs. P. Djian and H. Green (63).




DISCUSSION

The AP1 Sites AP1-1 and AP1-5 Enhance hINV Gene Expression-We have previously shown that 2.5 kilobase pairs of hINV gene upstream regulatory region are sufficient to drive tissue-specific expression in transgenic mice (40) . In the present study we have evaluated the ability of this region to drive transcription in keratinocytes. Sequential truncation of the upstream sequence results in a progressive loss of reporter gene activity. We have identified five AP1 consensus binding sites in this region. Two large drops in transcriptional activity are associated with the loss of DNA segments containing the AP1-1 and AP1-5 sites (Fig. 1). In contrast, the sites AP1-2, -3, and -4 make less of a contribution to the overall promoter activity. However, each deletion was quite large, potentially removing other unidentified transcription factor binding sites (see Fig. 1). We therefore confirmed by point mutation that AP1-1 and AP1-5 were required for optimal activity. An 80% reduction in transcriptional activity in keratinocytes was observed when AP1-1 and AP1-5 were simultaneously mutated, suggesting the these sites were necessary for hINV expression in keratinocytes (40) . Simultaneous mutation of both sites does not, however, completely abolish promoter activity, suggesting that other transcription factors mediate at least 20% of the activity.

hINV Promoter Is Silent in Fibroblasts

In contrast, the hINV promoter was found to be completely inactive in fibroblasts. This is consistent with the observation that no involucrin mRNA is detected in fibroblasts (12, 15, 40) (Fig. 9). Thus, although AP1-1 and AP1-5 are necessary for activity in keratinocytes, these sites are not sufficient to mediate transcriptional activation in fibroblasts. Possible explanations for the lack of activity of the hINV promoter in 3T3 cells is that 3T3 cells lack the necessary AP1 transcription factors and/or these factors are not appropriately phosphorylated. However, AP1-binding proteins have been described in fibroblasts (51) . Alternatively, some other factor may suppress hINV promoter transcription in 3T3 cells. Moreover, AP1 itself has been reported to suppress transcription in some systems (52) . Finally, a cofactor may be required for activation of hINV gene expression, expression of such a factor could be restricted to keratinocytes.

Activator Protein-1 Is Necessary for Differentiation-dependent Induction of hINV Expression

TPA has been shown to increase keratinocyte differentiation and to modulate gene expression in keratinocytes (50) . AP1 has been implicated in mediating the effects of phorbol esters in a variety of systems (20) . Our results show that TPA produces a 10-100-fold increase in AP1 binding to the hINV promoter AP1-1 and AP1-5 sites. This is consistent with a role for AP1 factors in mediating the differentiation-dependent increase in hINV gene expression. It was therefore important to determine whether the effects of TPA on hINV promoter activity were mediated by AP1. The point mutation experiment, shown in Fig. 8, suggests that the TPA response is partially mediated via the AP1-1 and AP1-5 binding sites. The fold induction is slightly reduced by mutation of AP1-1 or AP1-5 and further reduced by simultaneous inactivation of AP1-1 and AP1-5. These results suggest that the AP1-1 and AP1-5 sites cooperate to mediate one-half of the phorbol ester induction. This also implies that other transcription factors must participate in the induction events. The possibility that binding sites for these factors are localized in the hINV distal promoter region is currently being investigated. Identity of fos and jun Family Members Binding to AP1-1 and AP1-5-Gel mobility shift experiments demonstrate the specific binding of nuclear proteins to oligonucleotides containing the AP1-1 and AP1-5 sites. Competition for binding to radiolabeled AP1-1 or AP1-5 oligonucleotides was observed with oligonucleotides containing intact AP1-1 sites, but not with oligonucleotides containing mutant AP1 sites. In addition, oligonucleotides containing sites for other transcription factors (i.e. SP1, AP2, etc.) did not compete. Antibody supershift experiments using broad spectrum anti-fos and anti-jun antibodies (i.e. antibodies that react with all known family members) strongly suggest the participation of both fos and jun family proteins in the formation of the complexes which bind at the involucrin AP1-5 and AP1-1 sites. A more detailed supershift analysis using antibodies specific for each family member suggests that Fra-1 is the fos family contributor and junB and junD the jun family contributors. Fra-1 has been identified in heart and skeletal muscle (53, 54) , brain (55) , leukocytes (32) , fibroblasts (51), adipocytes (56) , the vascular system (57) , and in skeletal tissues (58) . This, however, is the first study to support a regulatory function for Fra-1 in keratinocytes. The 80% reduction in promoter activity in the absence of AP1-1 and AP1-5 suggests that Fra-1 may play a major role in the activation of hINV expression in the suprabasal epidermal layers. Immunohistological studies() indicate that Fra-1 is localized in the epidermal suprabasal layers. junB and junD have been implicated in the keratinocyte-specific expression of the human papillomavirus type 18 promoter (59) . Our results showing junB and junD binding to the hINV promoter suggest that these factors are important for hINV expression in keratinocytes.

Implications for Keratinocyte Differentiation Studies

In the present study we show that the AP1 transcription factor is necessary for expression of the hINV gene in epidermal keratinocytes. AP1 is particularly interesting in the broader context of a terminal differentiation program in which some genes are turned on while others are concurrently turned off, since it can act as both an activator and a repressor of transcription (52) . Extracellular calcium concentrations are higher in the upper than in the lower layers of the epidermis in vivo. In vitro, similar elevations of extracellular calcium cause keratinocytes to terminally differentiate and form envelopes (60, 61) . In keratinocytes, one intracellular response to increased extracellular calcium is the elevation of diacylglycerol levels and the activation of the protein kinase C pathway (61) and induction of terminal differentiation in tissue culture systems by the diacylglycerol analogue TPA most likely occurs by activation of protein kinase C. Protein kinase C is known to influence gene expression through AP1. In most systems, the activation of AP1 is a transient occurrence associated with mitogen stimulation (36) . In skin, however, the AP1 factor, c-fos, is constitutively expressed in the suprabasal layers (16) , suggesting it may have a role in regulating differentiation-specific gene expression. Our study suggests that hINV expression in keratinocytes is enhanced by the combined activity of several AP1 transcription factors, including Fra-1, junB, and junD and implies that these factors may be important for the differential regulation of expression of other keratinocyte genes.

In summary, our results indicate that the AP1-1 (distal promoter) and AP1-5 (proximal promoter) sites, (i) function as general enhancer elements, and (ii) mediate the differentiation-dependent increase in promoter activity in response to phorbol ester.

  
Table: Sequence of hINV AP1 sites and oligonucleotides

AP1-1, -2, -3, -4, and -5 are the AP1 binding sites from the hINV gene sequence with flanking sequence (see positions in Fig. 1). AP1-1m, AP1-2m and AP1-5m are mutants of AP1-1, -2, and -5 in which the T at position 5 of the consensus sequence is changed to C (underlined in each oligonucleotide). AP1c, SP1c, and AP2c contain the AP1, SP1, and AP2 consensus binding sites flanked by random sequence (Promega). The length of each double stranded oligonucleotide is shown at the right, the sequence of the putative AP1, SP1, or AP2 binding sites are indicated in bold.



FOOTNOTES

*
The work was supported in part by a grant from the National Institutes of Health (to R. L. E.) and utilized the facilities of the Skin Diseases Research Center of Northeast Ohio, supported by National Institutes of Health Grant AR39750. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by Cell Physiology Training Program Grant DK07678 from the National Institutes of Health and the Department of Orthopedics at Case Western Reserve University.

Recipient of the Albert Kligman Research Fellowship from the Dermatology Foundation sponsored by the Dermatological Division of Ortho Pharmaceutical Corp.

**
To whom correspondence should be addressed: Dept. of Physiology/Biophysics, Rm. E532, Case Western Reserve University School of Medicine, 2109 Adelbert Rd., Cleveland, OH 44106-4970. Tel.: 216-368-5530; Fax: 216-368-5586.

The abbreviations used are: hINV, human involucrin; KSFM, serum-free keratinocyte media; TPA, 12-O-tetradecanoylphorbol-13-acetate.

J. F. Welter and R. L. Eckert, unpublished data.


REFERENCES
  1. Eckert, R. L.(1989) Physiol. Rev. 69, 1316-1345 [Free Full Text]
  2. Rice, R. H., and Green, H.(1977) Cell 11, 417-422 [Medline] [Order article via Infotrieve]
  3. Yaffe, M. B., Murthy, S., and Eckert, R. L.(1993) J. Invest. Dermatol. 100, 3-9 [Abstract]
  4. Rice, R. H., and Green, H.(1979) Cell 18, 681-694 [Medline] [Order article via Infotrieve]
  5. Watt, F. M., and Green, H.(1981) J. Cell Biol. 90, 738-742 [Abstract]
  6. Thacher, S. M., and Rice, R. H.(1985) Cell 40, 685-695 [Medline] [Order article via Infotrieve]
  7. Steven, A. C., Bisher, M. E., Roop, D. R., and Steinert, P. M.(1990) J. Struct. Biol. 104, 150-162 [Medline] [Order article via Infotrieve]
  8. Yoneda, K., McBride, O. W., Korge, B. P., Kim, I. G., and Steinert, P. M.(1992) J. Dermatol. 19, 761-764 [Medline] [Order article via Infotrieve]
  9. Lee, S. C., Kim, I. G., Marekov, L. N., O'Keefe, E. J., Parry, D. A., and Steinert, P. M.(1993) J. Biol. Chem. 268, 12164-12176 [Abstract/Free Full Text]
  10. Banks-Schlegel, S., and Green, H.(1981) J. Cell Biol. 90, 732-737 [Abstract]
  11. Warhol, M. J., Antonioli, D. A., Pinkus, G. S., Burke, L., and Rice, R. H.(1982) Human Pathol. 13, 1095-1099 [Medline] [Order article via Infotrieve]
  12. Eckert, R. L., and Green, H.(1986) Cell 46, 583-589 [Medline] [Order article via Infotrieve]
  13. Yaffe, M. B., Beegen, H., and Eckert, R. L.(1992) J. Biol. Chem. 267, 12233-12238 [Abstract/Free Full Text]
  14. Eckert, R. L., Yaffe, M. B., Crish, J. F., Murthy, S., Rorke, E. A., and Welter, J. F.(1993) J. Invest. Dermatol. 100, 613-617 [Abstract]
  15. Murthy, S., Crish, J. F., Zaim, T. M., and Eckert, R. L.(1993) J. Struct. Biol. 111, 68-76 [CrossRef][Medline] [Order article via Infotrieve]
  16. Fisher, C., Byers, M. R., Iadarola, M. J., and Powers, E. A.(1991) Development 111, 253-258 [Abstract]
  17. Dlugosz, A. A., and Yuspa, S. H.(1993) J. Cell Biol. 120, 217-225 [Abstract]
  18. Basset-Seguin, N., Demoly, P., Moles, J. P., Tesnieres, A., Gauthier-Rouviere, C., Richard, S., Blanchard, J. M., and Guilhou, J. J.(1994) Oncogene 9, 765-771 [Medline] [Order article via Infotrieve]
  19. Lee, W., Haslinger, A., Karin, M., and Tijian, R.(1987) Nature 325, 368-372 [CrossRef][Medline] [Order article via Infotrieve]
  20. Lee, W., Mitchell, P., and Tjian, R.(1987) Cell 49, 741-752 [Medline] [Order article via Infotrieve]
  21. Angel, P., Imagawa, M., Chiu, R., Stein, B., Imbra, R. J., Rahmsdorf, H. J., Jonat, C., Herrlich, P., and Karin, M.(1987) Cell 49, 729-739 [Medline] [Order article via Infotrieve]
  22. Ransone, L. J., and Verma, I. M.(1990) Annu. Rev. Cell Biol. 6, 539-557 [CrossRef]
  23. Ryder, K., and Nathans, D.(1988) Proc. Natl. Acad. Sci. U. S. A. 85,8464-8467 [Abstract]
  24. Ryder, K., Lanahan, A., Perez-Albuerne, E., and Nathans, D.(1989) Proc. Natl. Acad. Sci. U. S. A. 86, 1500-1503 [Abstract]
  25. Hirai, S. I., Ryseck, R. P., Mechta, F., Bravo, R., and Yaniv, M. (1989) EMBO J. 8, 1433-1439 [Abstract]
  26. Zerial, M., Toschi, L., Ryseck, R. P., Schuermann, M., Muller, R., and Bravo, R.(1989) EMBO J. 8, 805-813 [Abstract]
  27. Cohen, D. R., and Curran, T.(1989) Crit. Rev. Oncog. 1, 65-88 [Medline] [Order article via Infotrieve]
  28. Cohen, D. R., Ferreira, P. C., Gentz, R., Franza, B. R., Jr., and Curran, T.(1989) Genes & Dev. 3, 173-184
  29. Nishina, H., Sato, H., Suzuki, T., Sato, M., and Iba, H.(1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3619-3623 [Abstract]
  30. Suzuki, T., Okuno, H., Yoshida, T., Endo, T., Nishina, H., and Iba, H. (1991) Nucleic Acids Res. 19, 5537-5542 [Abstract]
  31. Kovary, K., and Bravo, R.(1992) Mol. Cell. Biol. 12, 5015-5023 [Abstract]
  32. Boise, L. H., Petryniak, B., Mao, X., June, C. H., Wang, C. Y., Lindsten, T., Bravo, R., Kovary, K., Leiden, J. M., and Thompson, C. B. (1993) Mol. Cell. Biol. 13, 1911-1919 [Abstract]
  33. Redner, R. L., Lee, A. W., Osawa, G. A., and Nienhuis, A. W.(1992) Oncogene 7, 43-50 [Medline] [Order article via Infotrieve]
  34. Candeliere, G. A., Prud'homme, J., and St-Arnaud, R.(1991) Mol. Endocrinol. 5, 1780-1788 [Abstract]
  35. Castellazzi, M., Spyrou, G., La-Vista, N., Dangy, J. P., Piu, F., Yaniv, M., and Brun, G.(1991) Proc. Natl. Acad. Sci. U. S. A. 88, 8890-8894 [Abstract]
  36. Muller, R., Tremblay, J. M., Adamson, E. D., and Verma, I. M.(1983) Nature 304, 454-456 [Medline] [Order article via Infotrieve]
  37. Rothnagel, J. A., Greenhalgh, D. A., Gagne, T. A., Longley, M. A., and Roop, D. R.(1993) J. Invest. Dermatol. 101, 506-513 [Abstract]
  38. Huff, C. A., Yuspa, S. H., and Rosenthal, D.(1993) J. Biol. Chem. 268, 377-384 [Abstract/Free Full Text]
  39. Takahashi, H., and Iizuka, H.(1993) J. Invest. Dermatol. 100, 10-15 [Abstract]
  40. Crish, J. F., Howard, J. M., Zaim, T. M., Murthy, S., and Eckert, R. L. (1993) Differentiation 53, 191-200 [Medline] [Order article via Infotrieve]
  41. Prestridge, D. S.(1991) Comput. Appl. Biosci. 7, 203-206 [Abstract]
  42. Kay, R., and McPherson, J.(1987) Nucleic Acids Res. 15, 2778 [Medline] [Order article via Infotrieve]
  43. Risse, G., Jooss, K., Neuberg, M., Bruller, H. J., and Muller, R. (1989) EMBO J. 8, 3825-3832 [Abstract]
  44. Longley, J., Ding, T. G., Cuono, C., Durden, F., Crooks, C., Hufeisen, S., Eckert, R., and Wood, G. S.(1991) J. Invest. Dermatol. 97, 974-979 [Abstract]
  45. Rheinwald, J. G., and Green, H.(1975) Cell 6, 331-344 [Medline] [Order article via Infotrieve]
  46. Wirth, P. J., Yuspa, S. H., Thorgeirsson, S. S., and Hennings, H. (1987) Cancer Res. 47, 2831-2838 [Abstract]
  47. Dignam, J. D., Lebovitz, R. M., and Roeder, R. G.(1983) Nucleic Acids Res. 11, 1475-1489 [Abstract]
  48. Schreiber, E., Matthias, P., and Schaffner, W.(1989) Nucleic Acids Res. 17, 6419 [Medline] [Order article via Infotrieve]
  49. Distel, R. J., Ro, H. S., Rosen, B. S., Groves, D. L., and Spiegelman, B. M.(1987) Cell 49, 835-844 [Medline] [Order article via Infotrieve]
  50. Younus, J., and Gilchrest, B. A.(1992) J. Cell. Physiol. 152, 232-239 [Medline] [Order article via Infotrieve]
  51. Braselmann, S., Graninger, P., and Busslinger, M.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1657-1661 [Abstract]
  52. Takimoto, M., Quinn, J. P., Farina, A. R., Staudt, L. M., and Levens, D.(1989) J. Biol. Chem. 264, 8992-8999 [Abstract/Free Full Text]
  53. Hannan, R. D., Stennard, F. A., and West, A. K.(1993) J. Mol. Cell. Cardiol. 25, 1137-1148 [CrossRef][Medline] [Order article via Infotrieve]
  54. Park, K., Chung, M., and Kim, S. J.(1992) J. Biol. Chem. 267, 10810-10815 [Abstract/Free Full Text]
  55. Persico, A. M., Schindler, C. W., O'Hara, B. F., Brannock, M. T., and Uhl, G. R.(1993) Brain. Res. Mol. Brain. Res. 20, 91-100 [Medline] [Order article via Infotrieve]
  56. Stephens, J. M., Butts, M., Stone, R., Pekala, P. H., and Bernlohr, D. A.(1993) Mol. Cell. Biochem. 123, 63-71 [Medline] [Order article via Infotrieve]
  57. Miano, J. M., Vlasic, N., Tota, R. R., and Stemerman, M. B.(1993) Am. J. Pathol. 142, 715-724 [Abstract]
  58. Wang, Z. Q., Grigoriadis, A. E., and Wagner, E. F.(1993) J. Bone Miner. Res. 8, 839-847 [Medline] [Order article via Infotrieve]
  59. Thierry, F., Spyrou, G., Yaniv, M., and Howley, P.(1992) J. Virol. 66, 3740-3748 [Abstract]
  60. Menon, G. K., Grayson, S., and Elias, P. M.(1985) J. Invest. Dermatol. 84, 508-512 [Abstract]
  61. Lee, E., and Yuspa, S. H.(1991) Carcinogenesis 12, 1651-1658 [Abstract]
  62. Gilfix, B. M., and Eckert, R. L.(1985) J. Biol. Chem. 260, 14026-14029 [Abstract/Free Full Text]
  63. Djian, P., Phillips, M., Easley, K., Huang, E., Simon, M., Rice, R. H., and Green, H.(1993) Mol. Biol. Evol. 10, 1136-1149 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.