©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interferon--mediated Inactivation of Transcription of the 230-kDa Bullous Pemphigoid Antigen Gene (BPAG1) Provides Novel Insight into Keratinocyte Differentiation (*)

(Received for publication, June 6, 1994)

Katsuto Tamai Kehua Li (§) Stephanie Silos Lidia Rudnicka Takashi Hashimoto (1) Takeji Nishikawa (1) Jouni Uitto (¶)

From the Departments of Dermatology and of Biochemistry and Molecular Biology, Jefferson Medical College, and the Section of Molecular Dermatology, Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 and the Department of Dermatology, Keio University School of Medicine, Tokyo 160, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Interferon- (IFN-) has been shown to regulate epidermal keratinocyte growth and differentiation. In this study, we examined the effects of recombinant human IFN- on the expression of the gene encoding the 230-kDa bullous pemphigoid antigen (BPAG1), a marker of the mitotic basal cell phenotype in the epidermis. Northern analysis revealed a dose- and time-dependent suppression of BPAG1 expression by IFN- in cultured human keratinocytes from several different donors, and incubation of the cells with IFN- in the presence of cycloheximide demonstrated that this effect required ongoing protein synthesis. The inhibition of BPAG1 gene expression was also demonstrated at the protein level by indirect immunofluorescence using a monoclonal antibody recognizing the human 230-kDa bullous pemphigoid antigen. Transient transfections of cultured keratinocytes with BPAG1 promoter-chloramphenicol acetyltransferase reporter gene plasmids indicated marked suppression of the promoter activity by IFN-, and deletion constructs were able to identify a defined region containing the responsive element (IFN- inhibitory element). Reduced transcription of the BPAG1 gene by IFN- was also demonstrated by in vitro nuclear run-on assays. These data, which indicate inactivation of transcription of a basal keratinocyte-specific gene (BPAG1) by IFN-, provide novel insight into the mechanisms of IFN--mediated keratinocyte gene regulation and epidermal differentiation in inflammatory diseases.


INTRODUCTION

Terminal differentiation of epidermal keratinocytes from the proliferative basal phenotype to formation of the cornified envelope involves activation and inactivation of a variety of genes at different levels of the epidermis. One of the markers of the mitotic basal cell phenotype is the expression of bullous pemphigoid antigens (BPA), (^1)two hemidesmosomal proteins that were initially recognized as autoantigens in a blistering skin disease, bullous pemphigoid(1, 2, 3) . It is now known that there are at least two bullous pemphigoid antigens, the 230- and 180-kDa BPA; the corresponding genes are known as BPAG1 and BPAG2, respectively(4, 5) . Recent cloning of BPAG1 and BPAG2 complementary and genomic DNA sequences has shown that these two proteins are clearly distinct gene products(6, 7, 8, 9, 10, 11, 12, 13) .

Recent studies have demonstrated that interferon- (IFN-) regulates keratinocyte growth and differentiation(14) . This cytokine, a product of activated T cells and natural killer cells(15) , has been shown to affect the expression of a variety of genes(16) . In general, the IFN- effects are up-regulatory, and the corresponding transcriptional mechanisms have been elucidated in the case of several genes(16, 17, 18) . In selected cases, as for example in the case of type I collagen, IFN- down-regulates the expression of the corresponding genes, COL1A1 and COL1A2, but the mechanism is clearly post-transcriptional(17) .

IFN- has two well documented effects on epidermal cell biology. First, this cytokine induces the expression of intercellular adhesion molecule-1, which serves as the binding molecule for lymphocyte function-associated antigen-1, mediating lymphocyte-keratinocyte interactions(19) . Furthermore, IFN- induces the expression of HLA-DR on keratinocytes(20, 21) . Thus, the effects of IFN- play a key role in epidermal immune responses. Second, IFN- has direct inhibitory effects on keratinocyte growth and induces terminal differentiation(14, 22) . Specifically, IFN- up-regulates the expression of differentiation-specific genes, including transglutaminase type I and a variety of proteins involved in the formation of the cornified envelope(14) .

Since down-regulation of BPA gene expression is one of the earliest events during epidermal differentiation, in this study, we examined the effects of IFN- on BPAG1 and BPAG2 gene expression.


MATERIALS AND METHODS

Keratinocyte Cultures

Human epidermal keratinocyte cultures were established from skin left over from cosmetic surgery procedures of adult subjects. The cultures were maintained in serum-free, low calcium (0.15 mM) keratinocyte growth medium supplemented with epidermal growth factor, hydrocortisone, insulin, and bovine pituitary extract (KGM, Clonetics Corp., San Diego, CA). Cultures were passaged by trypsinization and studied in passage 2. Some cultures were incubated with recombinant human IFN-, which was purchased from Boehringer Mannheim. The specific activity of IFN- was >2 times 10^7 units/mg.

Northern Analyses

Total RNA was isolated from cultured keratinocytes by a single-step extraction method(23) . For Northern hybridizations, total RNA (30 µg/lane) was fractionated on 1.0% agarose gels and transferred to nitrocellulose filters(24) . The filters were prehybridized and then hybridized with a 2.3-kb human BPAG1 cDNA (25) radioactively labeled with [alpha-P]GTP and [alpha-P]CTP by nick translation(26) . The filters were washed to a final stringency of 0.5 times SSC, 0.1% SDS at 65 °C. The filters were then exposed to x-ray films (X-Omat, Eastman Kodak Co.). After obtaining autoradiograms at different levels of exposure, the BPAG1 cDNA was removed by incubating the filters in 0.1 times SSC, 0.5% SDS at 99 °C. The filters were prehybridized and then successively rehybridized with a 1.0-kb human BPAG2 cDNA (10) and with a 1.3-kb human cDNA for glyceraldehyde-3-phosphate dehydrogenase, a ubiquitously expressed housekeeping gene(27) .

The intensity of the autoradiographic bands was quantitated by scanning densitometry of the x-ray films at 633 nm, and the BPAG1 and BPAG2 mRNA steady-state levels were corrected by the levels of glyceraldehyde-3-phosphate dehydrogenase mRNA transcripts in the same RNA preparations.

Transient Transfections of Keratinocytes with the BPAG1 Promoter-CAT Construct

The human BPAG1 promoter-CAT reporter gene plasmid, pBP2.6CAT, containing the human BPAG1 promoter region extending from positions -2536 to -1 (in relation to the transcription initiation site) (28) was used for transient transfections of cultured keratinocytes. In addition, 14 separate 5`-deletion constructs, the 5`-end of the promoter spanning from positions -2536 to -27, were developed and used for parallel transfections. The cells were cotransfected with a Rous sarcoma virus-beta-galactosidase construct, which was used as an internal control of transfection efficiency. The transfections were performed with a commercial kit (DOTAP, Boehringer Mannheim), and IFN- at varying concentrations was added to some of the cultures at the time of transfection. In parallel transfections, a thymidine kinase promoter-CAT construct (29) was used for transfections with and without IFN- (1000 units/ml). The transfection medium was removed at 24 h of incubation and replaced with fresh medium containing the same concentrations of IFN-. After a total of 48 h of incubation, the CAT activity in the cell cultures was determined by incubation of cell extracts with [^14C]chloramphenicol, followed by separation of its acetylated and non-acetylated forms by thin-layer chromatography, as described elsewhere(28, 30) . The promoter activity was determined by counting the radioactivity in the acetylated forms of chloramphenicol as a percent of total radioactivity and was corrected for the beta-galactosidase activity in the same cell extract.

In Vitro Nuclear Run-on Transcription Assay

Nuclei were prepared from cultured keratinocytes, and in vitro synthesis of radiolabeled nascent RNA was performed as described elsewhere(31) . The RNA transcripts were purified and hybridized with BPAG1 cDNA immobilized on nitrocellulose filters. Parallel hybridizations were performed with human ribosomal 7 S cDNA as well as with the pBluescript vector. The hybridization and washing conditions were as described before(31) . Radioactivity in the RNA-cDNA hybrids was visualized by autoradiography and quantified by scanning densitometry.

Indirect Immunofluorescence

For immunostaining, keratinocytes were cultured on glass chamber slides with and without IFN- for 24 or 48 h. The cells were then fixed in cold (-20 °C) ethanol, rinsed with Tris-buffered saline (pH 7.6), and preincubated for 60 min with Tris-buffered saline containing 1% bovine serum albumin. The samples were then exposed to a human monoclonal anti-human 230-kDa BPA monoclonal antibody (32) overnight at 4 °C. The slides were washed in Tris-buffered saline for 60 min and incubated with tetramethylrhodamine isothiocyanate-conjugated anti-human IgG antibody (Miles, Inc.). Controls incubated without the primary antibody gave essentially negative staining. After a 60-min incubation at room temperature, the slides were washed with Tris-buffered saline for 60 min, rinsed with distilled water, mounted, and examined with a fluorescent microscope. The immunosignal was semiquantitatively assessed by spectrophotometric scanning densitometry (Densitron Model PAN, Jookoo Co., Japan).


RESULTS

Selective Down-regulation of BPAG1 by Interferon-

To examine the effects of IFN- on BPAG1 and BPAG2 gene expression, cultured human epidermal keratinocytes were incubated with IFN- at varying concentrations, and the corresponding mRNA levels were determined by Northern hybridizations. In control cultures, characteristic mRNA transcripts for BPAG1 and BPAG2, 9.0 and 6.0 kb in size, respectively, were detected. Further analyses indicated that the BPAG1 gene was markedly down-regulated with as little as 10 units/ml IFN- after correction of the mRNA steady-state levels by the corresponding levels of glyceraldehyde-3-phosphate dehydrogenase (Fig. 1). The inhibition of BPAG1 gene expression was dose-dependent, and the inhibition was 90% in the presence of 1000 units/ml IFN- (Fig. 1B). In contrast, no change in BPAG2 gene expression was noted in the presence of 10 or 100 units/ml IFN-, and only a slight (20%) inhibition was noted in the presence of 1000 units/ml (Fig. 1B). The inhibition of BPAG1 gene expression, as determined at the mRNA steady-state level, in the presence of 100 units/ml IFN-, was evident at 3 h of incubation, and maximal inhibition was noted at 12 h (Fig. 2). Again, rehybridization of the same filter with a BPAG2 cDNA indicated a relatively small (20%) and transient inhibition (Fig. 2B).


Figure 1: Effects of IFN- on BPAG1 and BPAG2 gene expression in cultured human keratinocytes. The cells were incubated with recombinant human IFN- at concentrations varying from 0 to 1000 units/ml for 24 h. Total RNA was then isolated, and Northern analyses with BPAG1-, BPAG2-, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific cDNAs were performed as described under ``Materials and Methods.'' A, autoradiograms of the Northern filters; B, quantitation of relative mRNA levels by scanning densitometry. The values are expressed as a percent of control cultures incubated in parallel without IFN- (100%).




Figure 2: Time-dependent inhibition of BPAG1 gene expression by IFN-. Cultured keratinocytes were incubated in the presence of 100 units/ml IFN- for the time periods indicated, and BPAG1 and BPAG2 mRNA steady-state levels were determined by Northern hybridizations as described in the legend to Fig. 1. A, autoradiograms of the Northern filters; B, quantitation of the mRNA levels by scanning densitometry. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.



To examine whether the IFN--elicited down-regulation of the BPAG1 gene was dependent of ongoing protein synthesis, similar inhibition studies were performed in the presence and absence of cycloheximide (10 µg/ml). As indicated above, incubation of cultured keratinocytes with IFN- (1000 units/ml) for 24 h in the absence of cycloheximide resulted in essentially complete reduction of BPAG1 mRNA steady-state levels (Fig. 3). Incubation of parallel cultures with cycloheximide alone resulted in 50% inhibition of the BPAG1 mRNA steady-state level after correction for the glyceraldehyde-3-phosphate dehydrogenase mRNA levels, which were slightly increased in the presence of cycloheximide. However, the addition of IFN- failed to down-regulate the BPAG1 mRNA levels in the presence of cycloheximide (Fig. 3). These observations suggest that the effect of IFN- on BPAG1 gene expression requires ongoing protein synthesis, possibly induction of trans-acting regulatory proteins.


Figure 3: Demonstration that IFN--elicited down-regulation of BPAG1 gene expression at the mRNA level is dependent on ongoing protein synthesis. Keratinocyte cultures were incubated in the presence (+) or absence(-) of IFN- (1000 units/ml) and/or cycloheximide (CHX; 10 µg/ml) for 24 h. Northern hybridizations with BPAG1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs were performed as described in the legend to Fig. 1.



Evidence for Transcriptional Regulation of BPAG1 Gene Expression by IFN-

To examine the potential mechanisms that might be responsible for IFN--elicited down-regulation of BPAG1 gene expression as detected at the mRNA level, transient transfections with a BPAG1 promoter-CAT construct were performed in cultured keratinocytes. This construct consists of 2.6 kb of 5`-flanking DNA of the BPAG1 gene, a segment that has been previously shown to contain cis-elements conferring tissue-specific expression of the gene(28, 33) . Transfection of control keratinocytes with this plasmid construct revealed strong expression of the promoter, as detected by CAT assay (Fig. 4A). The addition of IFN- to the culture medium resulted in a marked inhibition of the promoter activity, and in fact, the promoter activity in cultures incubated with 10 units/ml IFN- for 48 h was only 6% of that noted in control cultures transfected without IFN- in parallel. Further increase of IFN- concentration to 100 or 1000 units/ml resulted in 98% inhibition of the promoter activity (Fig. 4A). At the same time, parallel transfections with a thymidine kinase promoter-CAT construct indicated that IFN- at concentrations as high as 1000 units/ml did not affect the CAT activity (Fig. 4B), indicating that IFN- did not alter the stability of the CAT mRNA transcript or CAT protein. It should be noted that the BPAG1 promoter-CAT construct was cotransfected in all experiments with a Rous sarcoma virus-beta-galactosidase construct, and the values for CAT activity were corrected for beta-galactosidase activity in the same cell extracts. Consequently, the marked down-regulation of BPAG1 promoter activity by IFN- cannot be explained by altered transfection efficiency. Thus, these observations suggest that the relative reduction of BPAG1 mRNA levels by IFN- takes place primarily at the transcriptional level of gene expression.


Figure 4: Selective inhibition of BPAG1 promoter activity in transient transfections of cultured keratinocytes. Human epidermal keratinocytes were transfected with the pBP2.6CAT construct, which consists of human BPAG1 gene 5`-flanking DNA sequences extending from positions -2536 to -1 linked to the CAT reporter gene. The keratinocytes were cotransfected with Rous sarcoma virus-beta-galactosidase, which was used as an internal control of transfection efficiency. The transfected cells were incubated with IFN- at concentrations varying from 0 to 1000 units/ml (lanes 1-4 in A). Parallel transfections were performed with a thymidine kinase promoter-CAT construct in the absence or presence of 1000 units/ml IFN- (lanes1 and 2 in B). After 48 h of incubation, the CAT activity was determined as described under ``Materials and Methods.'' The relative CAT activity was determined by counting the radioactivity in the acetylated forms of radioactive chloramphenicol (AC) as a percent of the total radioactivity in the acetylated and non-acetylated (C) forms of [^14C]chloramphenicol used as substrate.



To identify the region within the 2.6-kb BPAG1 promoter that is responsive to IFN-, eight additional 5`-deletion constructs were similarly tested in transient transfections. Each construct was transfected in parallel to duplicate cultures, with or without concomitant treatment with IFN- (100 units/ml) (Fig. 5). The results indicated that seven of the constructs, with their 5`-ends at positions -1806 to -216, which demonstrated significant CAT activity, were responsive to IFN-. The construct extending from positions -176 to -1 had too low level of expression to evaluate IFN- response. Nevertheless, the putative IFN- inhibitory element resides within 0.2 kb of the BPAG1 promoter region.


Figure 5: Transient transfections with BPAG1 promoter 5`-deletion CAT constructs. Cultured keratinocytes were transfected with constructs containing the BPAG1 promoter region with their 5`-ends at the positions indicated. The cultures were then incubated without(-) or with (+) IFN- (100 units/ml), and the CAT activity was determined as described in the legend to Fig. 4.



To further examine the modulation of BPAG1 gene expression by IFN- at the transcriptional level, in vitro nuclear run-on transcription assays were performed. Quantitation of the BPAG1 RNA transcripts in nuclei isolated from keratinocytes, incubated with or without IFN- (1000 units/ml), indicated a 68% reduction by IFN- after correction for ribosomal 7 S RNA transcripts in the same incubation (Fig. 6). No hybridization was noted with the pBluescript vector as a negative control (Fig. 6).


Figure 6: In vitro nuclear run-on transcription analysis. Isolated human epidermal keratinocyte nuclei were incubated with [P]UTP, and radiolabeled RNA transcripts were isolated as described under ``Materials and Methods'' and elsewhere(31) . Human BPAG1 and ribosomal 7 S unlabeled cDNAs, as well as the pBluescript vector (BS), 5 µg each, were immobilized on nitrocellulose filters. The filters were prehybridized and then hybridized with radiolabeled RNAs. Left, autoradiogram from hybridization with RNA from control cell nuclei; right, nuclei from cells incubated with IFN- (1000 units/ml) for 24 h.



Inhibition of BPAG1 Gene Expression in Cultured Keratinocytes by IFN- as Determined at the Protein Level

To demonstrate that the reduced mRNA levels for the 230-kDa BPA are also accompanied by reduction at the protein level of gene expression, keratinocyte cultures were incubated with and without IFN- (1000 units/ml). The presence of the 230-kDa BPA was then determined by indirect immunofluorescence utilizing a monoclonal anti-human BPA antibody(32) . The results suggested marked reduction in the immunosignal at 24 h of incubation (Fig. 7). Densitometric scanning of the immunosignal indicated 48 and 56% reduction in the epitope levels in cells incubated with IFN- for 24 and 48 h, respectively.


Figure 7: Demonstration of reduced 230-kDa BPA protein levels in human keratinocytes incubated with IFN-. Confluent cultures of epidermal keratinocytes were incubated for 0 or 24 h with 1000 units/ml IFN-. The presence of the 230-kDa BPA protein was determined by indirect immunofluorescence with a monoclonal antibody.




DISCUSSION

In this study, we have demonstrated that IFN- effectively reduces the expression of the 230-kDa BPA in cultured keratinocytes as detected both at the mRNA and protein levels. This novel finding contrasts with the induction of a variety of keratinocyte gene products by IFN-, including intercellular adhesion molecule-1 and HLA-DR(19, 20) . Furthermore, IFN- is an up-regulatory cytokine for the expression of a variety of genes at the transcriptional level, and specific IFN--responsive elements, such as the IFN-stimulated response element and -activated site (see below), have been identified in the promoter regions of these genes(16, 18) . Mechanistically, the induction of transcription by IFN- involves activation of a latent cytoplasmic factor, -activated factor (GAF), by tyrosine phosphorylation(34, 35) . Activated GAF is then translocated into the nucleus and binds to a cis-element, the -activated site, first recognized in the gene encoding a guanylate-binding protein(34, 35, 36) . The GAF protein was subsequently shown to be identical to a 91-kDa DNA-binding protein, one of the four components of interferon-stimulated gene factor-3, a transcription factor previously shown to be activated by IFN-alpha(35) . The interferon-stimulated gene factor-3 complex then recognizes the interferon-stimulated response element present in a variety of IFN-alpha-inducible promoters(37, 38, 39, 40) . The phosphorylation of GAF in response to IFN- has been shown to be mediated by kinases known as the Jak family of protein-tyrosine kinases(41, 42) . In particular, Jak1 is activated in response to both IFN-alpha and IFN-, while Jak2 responds exclusively to IFN-(43) . Thus, these kinases are required for nuclear signaling induced by IFN-.

The reduced BPAG1 mRNA steady-state levels, as demonstrated in this study, result from reduced transcriptional activity of the gene, as shown by transient transfection experiments and nuclear transcription assays. The mechanisms of this down-regulation of gene expression are not clear, but they are unlikely to involve direct binding of GAF or interferon-stimulated gene factor-3 to DNA since ongoing protein synthesis was required for the IFN- effect, as demonstrated by cycloheximide experiments. Previously, IFN- has been shown to abrogate lipopolysaccharide-mediated enhancement of tumor necrosis factor receptor expression in murine peritoneal macrophages(44) . This effect also took place at the transcriptional level, but the mechanism appears to be different from our observations since the IFN- effect was independent of ongoing protein synthesis(44) .

The use of 5`-deletion constructs in transient transfections indicated that the putative IFN- inhibitory element resides within 0.2 kb of the upstream promoter region of the BPAG1 gene. Examination of this sequence identified keratinocyte-responsive element-3, which has been recently shown to confer keratinocyte-specific expression to the BPAG1 gene(45) . However, as indicated above, the keratinocyte-responsive element-3 sequence, 5`-CAAATATTTG-3` at positions -213 to -204, has no similarity to previously published IFN--responsive elements. Specifically, careful examination of the BPAG1 promoter region between positions -216 and -1, which was shown to contain the putative IFN- inhibitory element, did not reveal a nucleotide sequence reminiscent of the -activated site consensus sequence, TTNCNNNAAA (36) , or of the IFN-stimulated response element, (G/A)GGAAAN(N)GAAACT (18) .

The observations presented in this study provide novel insight into the mechanisms of keratinocyte gene expression, indicating that IFN- is a potential inducer of epidermal differentiation as reflected by down-regulation of a basal cell-specific gene, BPAG1. The transcriptional down-regulation of the BPAG1 gene was selective in that the expression of another hemidesmosomal gene, BPAG2, was not affected by IFN- as determined at the mRNA steady-state level. Since BPAG2 is also expressed exclusively in the basal keratinocytes within the epidermis, differential mechanisms are required for down-regulation of BPAG1 and BPAG2 genes at the suprabasilar level in vivo. These observations attest to the complexity of epidermal differentiation under physiological conditions.


FOOTNOTES

*
This work was supported in part by Grants PO1-AR38923 and T32-AR7561 from the National Institutes of Health and by the Dermatology Foundation. 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.

§
Recipient of a Dermatology Foundation research fellowship.

To whom correspondence should be addressed: Dept. of Dermatology, Rm. 450, Jefferson Medical College, 233 S. 10th St., Philadelphia, PA 19107. Tel: 215-955-5785; Fax: 215-955-5788.

(^1)
The abbreviations used are: BPA, bullous pemphigoid antigens; IFN, interferon; kb, kilobase pair(s); CAT, chloramphenicol acetyltransferase; GAF, -activated factor.


ACKNOWLEDGEMENTS

We thank Tamara Alexander for expert secretarial help and Lin Lin for skillful technical assistance. Dr. James W. Fox IV and Dr. John H. Moore, Jr. kindly provided tissue for cell cultures. The human BPAG2 cDNA was kindly provided by Dr. George Giudice (Medical College of Wisconsin).


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