Transglutaminase (TGase) ()enzymes are widespread in both plants and animals(1, 2, 3) . They catalyze the formation of an isodipeptide cross-link between the -NH side chain of a protein-bound lysine residue and the -amide side chain of a protein-bound glutamine residue, thereby forming an insoluble macromolecular aggregate that is used for a variety of cellular functions. To date, there are six known transglutaminase enzymes encoded in the human genome, and interestingly, three of them are active in the epidermis and its appendages. These include: the TGase 1 enzyme(4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) which can function as membrane-associated(8) , soluble full-length, and soluble proteolytically activated processed forms in the epidermis(17) ; the soluble, ``tissue'' TGase 2 enzyme (18, 19, 20, 21) ; and the soluble TGase 3 proenzyme, which also requires proteolytic activation(22, 23, 24, 25, 26, 27, 28, 29, 30, 31) .

The role of each of these enzymes in the fate of differentiating epidermal and hair follicle cells is not yet clear. The TGase 2 enzyme has been implicated in apoptosis, cell adhesion, and signal transduction(1, 2, 3, 32) . The TGase 1 and TGase 3 enzymes are thought to be required for the orderly assembly of specific structural proteins to form a specialized structure termed the cornified cell envelope which provides vital barrier functions for the organism(1, 2, 3, 33, 34, 35, 36, 37) . In addition, the TGase 3 enzyme is thought to be required for the cross-linking of the structural protein trichohyalin and the keratin intermediate filaments to form a rigid structure within the inner root sheath cells(38, 39, 40, 41, 42) , and thereby participate in shape determination of the developing hair cortical cells internal to the sheath structure (38, 39, 43) . In the medulla cells internal to the hair fiber, the trichohyalin is cross-linked to itself to form an insoluble vacuolized lattice-like structure which in turn is essential entrapment of air for thermal regulation in mammals(38, 43) . The involvement of TGases in cell envelope formation is supported by three types of observations. First, a large body of in vivo studies have documented that the TGases and the cell envelope structural proteins are co-expressed (reviewed in (1, 2, 3) and 33). Second, many in vitro cross-linking studies have shown that the cell envelope proteins or model peptides derived from them are efficiently used as substrates by these enzymes(33, 34, 44, 45) . Third, very recent studies have shown that mutations in the TGM1 gene that result in an inactive TGase 1 enzyme are the cause of lamellar ichthyosis, an autosomal recessive disorder of the cornification(46, 47, 48) . Thus it is quite likely that mutations in the TGM3 gene will also cause autosomal recessive ichthyosis-like diseases(46, 49) .

The ability of the three epidermally expressed TGases to cross-link the same substrates, although with different efficiencies(44) , creates a potential to provide for a missing TGase activity by appropriately modulating the expression of the two other enzymes. Hence, it is imperative to understand the mechanisms that control and modulate the expression of these TGase genes in the epidermis. TGM1, TGM2, and TGM3 genes have distinctly different patterns of expression. TGM2 is expressed in a variety of tissues (18, 19, 20, 21, 32) but in the epidermis it is largely restricted to proliferative basal keratinocytes(17) . The expression is induced both in vivo and in vitro by retinoids(50) , most probably through the retinoic acid receptor RAR-dependent signaling pathway(51) . The retinoic acid-induced TGase 2 activity is inhibited by the phorbol ester TPA and to a lesser extent by Ca(50) . TGM1 is expressed in all tissues of epithelial origin(4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) . Its transcription is regulated negatively by retinoids and is induced by TPA and calcium, primarily in suprabasal cells(28, 50, 52, 53) . TGM3, as far as is currently known, is expressed only during the last stages of terminal differentiation of the epidermis and epidermal appendage cell types such as the inner root sheath and medulla of the hair follicle(22, 23, 24, 25, 26, 27, 28, 29, 30, 31) . Its expression is initiated well after the transcription of the genes encoding the earlier differentiation markers, such as the keratin proteins K1 and K10, have been repressed (17) and approximately coincident with expression of the profilaggrin (54) and loricrin (55) genes. Although TGase 3 mRNA represents less than 2% of the TGase transcripts, the activated TGase 3 accounts for up to 75% of the total TGase activity in mammalian epidermis(17) . In submerged cultures of undifferentiated normal human epidermal keratinocytes (NHEK) the abundance of TGase 3 mRNA is greatly diminished compared to the foreskin epidermis. In contrast, TGase 1 and TGase 2 mRNA levels are increased. Induction of NHEK cell differentiation with Ca leads to an increase in the TGase 1 and TGase 3 mRNA expression, whereas TGase 2 mRNA is down-regulated(28) .

To date, very little is known about the recognition elements and the protein factors that regulate the transcription of these three TGase genes. In a recently published study on TGM 2 gene(56) , about 1.6 kb of the 5`-upstream region conferred low levels of constitutive activity that could not be modulated by retinoids, so that the sequences which control the high level and the retinoic acid inducibility of TGM2 transcription must be located elsewhere. In the case of the TGM1 gene, 0.82 kb of upstream sequences were found to induce expression in epithelial cells in the presence of TPA, which could be suppressed by retinoids or protein kinase C(57) . Co-transfection experiments indicated that c-jun and c-fos transcription factors were involved, presumably through an AP-1 site in this region, but none of the regulatory sequences have been defined functionally. In the case of the TGM3 gene, no data are available on the factors which control its expression.

The aim of this study has been to explore the mechanisms which regulate the expression of the human TGM3 gene. As an initial step, we have defined sequences in the vicinity of the mRNA start site which provide high promoter activity and which restrict expression to epithelial cells in vitro. Our data indicate that this epithelial-specific activity is provided by cooperative interactions between Sp1 and ets transcription factors.

MATERIALS AND METHODS

All recombinant DNA technology was done according to standard procedures(58, 59) .

Construction of Recombinant Clones

Two other commercial vectors were used for comparisons with the eight pCAT-Basic constructs. These were the pCAT-Promoter and pCAT-Control vectors (Promega), which are the same as the pCAT-Basic vector except that in the former the CAT gene is driven by the powerful SV40 promoter, and the latter contains SV40 early promoter and enhancer sequences.

In addition, we used a second heterologous promoter derived from the herpes simplex virus thymidine kinase gene. Sequences between -50 and +50 of the thymidine kinase gene from the pBLCAT2 vector (60) were used to construct a ptk-Promoter vector. TGM3 gene sequences between -126 and -73, derived from synthetic oligonucleotides, were inserted in front of this minimal promoter. The sequence of the resulting construct ptk-TGM3 was verified by sequencing.

Cell Cultures, Transfections, and Protein Assays

Transient transfections were performed in duplicate using Lipofectin reagent (Life Technologies) for NHEK, HeLa, A431, HepG2, and MCF-7 cells, or DOTAP (Boehringer Meinheim) for all other cells lines following the manufacturer's recommendations. Typically, 2-3 10 cells were plated in 6-well culture plates 16-20 h before transfection. Transfections were done when cultures reached 60-70% confluency. Transfection efficiencies were always monitored by use of a thymidine kinase -galactosidase construct (tk--gal) (Clontech, Palo Alto, CA). For Lipofectin transfections, cell cultures were washed once at 37 °C with phosphate-buffered saline, and then were preincubated for 30 min at 37 °C with either Keratinocyte-SFM (Life Technologies, Inc.) for NHEK cells, or Opti-MEMI media (Life Technologies, Inc.) for HeLa, A431, and HepG2 cells. For each well, 1.5 µg of reporter plasmids and 0.5 µg of tk--gal was mixed with 6 µg of Lipofectin, and incubated for 20 min at room temperature. The lipid/DNA mixture was then added into each well and incubated for 16-18 h in the case of NHEK and A431 cells, or 3-4 h in the case of HeLa and HepG2 cells. At the end of the transfection period the medium was replaced with the medium in which the cells normally grow, and for NHEK cells the concentration of the Ca in the KGM medium was adjusted to 1.2 mM. Cells were harvested 50-60 h post-transfection. For DOTAP transfections, the plasmids (1.5 µg of reporter constructs and 0.5 µg of tk--gal) were mixed with 14 µg of DOTAP in HEPES buffer solution (pH 7.3) and incubated for 20 min at room temperature. The transfections were carried out in the medium of each cell type for 16-18 h. The media were then replaced and cells cultured for another 50-60 h. Some NHEK cultures were co-transfected with the TGM3 -126/+10 sequences with the pECE vector or this vector containing mouse ets-2 cDNA (gift of Dr. Richard Maki, La Jolla Cancer Research Foundation). Similar co-transfection experiments used the human pRSV-Sp1 cDNA or the RSV-vector alone (gift of Dr. Robert Tjian, University of California, Berkeley).

Cellular extracts were prepared through at least three freeze-thaw cycles as described(61) . Aliquots were used for CAT assays, -gal assays, and total protein quantitation(62) . Cellular extracts of untransfected cells and of cells transfected with the pCAT-Basic vector alone without the TGM3 inserted sequences were used as negative controls, while the pCAT-Promoter and pCAT-Control vectors served as positive controls. CAT activities were determined (63) using chloramphenicol and [H]acetyl-CoA (DuPont NEN) as substrates. -Gal activity was assayed by using a commercial enzyme assay system (Promega). The values for CAT were normalized by protein content and -gal activity. The relative CAT values are the average of at least three independent experiments, each with duplicate samples.

Nuclear Extracts and Mobility Shift Assays

Mobility shift experiments were performed with 5 µg of nuclear extracts and 2-4 10 cpm (about 1 ng) of gel-purified 5` end-labeled double-stranded oligonucleotides containing the desired TGM3 sequences (see figures for sequences). Typically, the binding reactions were carried out in 20 µl containing 10 mM Tris-HCl (pH 7.5), 65 mM NaCl, 5 mM dithiothreitol, 5 mM MgCl, 0.05% Nonidet P-40, 10% glycerol, 1 mg/ml bovine serum albumin, and 25 µg/ml poly(dI-dC) as a carrier for 30 min at 4 °C. In competition experiments, a 100-fold molar excess of the cold competitor was preincubated with the extracts for 30 min at 4 °C before the labeled DNA fragment were added. Irrelevant control competitor oligonucleotides for consensus AP1 and AP2 sequences (Santa Cruz Biotechnology) were also used. Recombinant human Sp1 protein was from Promega and used as per the manufacturer's recommendations. The complexes were resolved on nondenaturing 6% polyacrylamide gels in 0.5 TBE buffer for 1 h at 14 V/cm, and viewed following overnight autoradiography.

RESULTS

The Proximal Promoter of the TGM3 Gene is Highly Active in NHEK Cells

Figure 1: Location of the proximal promoter of the TGM3 gene. The relative CAT activities of the several TGM3 constructs (A) were normalized with respect to the activity of the tk--gal construct and then expressed as a percentage of the activity of the pCAT-Promoter construct (B). The data are the averages of three or more independent experiments.

The Activity of TGM3 Proximal Promoter Is Specific to Stratified Squamous Epithelial Keratinocytes

DNA-Protein Interactions in the Proximal Promoter Region of the TGM3 Gene

In a pilot DNase I footprinting experiment using keratinocyte nuclear extract we detected two weak footprints on both DNA strands within the TGM3 promoter (data not shown). The protected region which extended from -73 to -102 contained an Sp1-like binding site. The second protected region was located between positions -111 and -128 and contained two direct copies of the sequence (T/C)TACAGG(C/A)A, which encompass ets-like recognition motifs.

Mobility shift experiments were aimed at characterizing in more detail these DNA-protein interactions. For each labeled probe the specificity of the binding was established in competition experiments with a 100-fold molar excess of either the corresponding unlabeled oligonucleotides (specific competitors), or of poly(dI-dC) or unrelated oligonucleotides as unspecific competitors.

Initially, a 46-base pair probe (-126 to -81, Fig. 2, lane 0) encompassing the putative Sp1 and ets binding sites was used. In the presence of a 100-fold molar excess of poly(dI-dC) (lane 1) or of the two irrelevant oligomers, consensus AP1 (lane 6) or consensus AP2 (lane 10) sequences, five retarded complexes were discerned. Specific competition with the unlabeled probe completely prevented the formation of complexes A, B, and C (lane 2). Competition experiments with wild type oligonucleotides derived from the probe revealed that complexes A and C involved the sequences of the DNase I protected region which encompassed the Sp1 recognition site (lanes 3 and 7), whereas complex B originated from interactions in the ets-like protected region which extended between -111 and -118 (lanes 4 and 8). The contribution of the putative Sp1- and ets-recognition motifs to the formation of these complexes was evaluated in competitive binding with mutant variants of the corresponding sequences. Mutations in the Sp1 binding motif (lanes 4 and 5) prevented competion for complexes A and C. Likewise, mutations in the core ets motif (lanes 7 and 9) strongly interfered with the ability of the mutant oligonucleotide to compete for complex B formation. However, a 100-fold molar excess of the oligonucleotides containing the intact Sp1-binding motif were not able to compete for ets binding (lanes 3 and 7) and, likewise, ets oligomers did not affect the formation of the Sp1 complexes (lanes 4 and 8). Two other bands marked with arrowheads were relatively resistant to self-competition (lane 2) and were formed both with double- and single-stranded oligonucleotides (data not shown), and have not been investigated further.

Figure 2: Gel mobility shift analysis of the TGM3 proximal promoter region with a probe encompassing the DNase I protected regions. The probe (-126 to -81, lane 0) was incubated with 5 µg of NHEK nuclear extract in the presence of a 100-fold molar excess of: poly(dI-dC) (lane 1); unlabeled probe (lane 2); and irrelevant consensus AP1 (lane 6), or consensus AP2 (lane 10) oligonucleotides. In lanes 3-5 and lanes 7-9, wild type and mutant variants of the probe sequence were used for competition. The letters in bold are the nucleotides of the ets and Sp1 motifs (boxed) that were mutated. In the oligonucleotides listed, dots represent unchanged nucleotides. Mutated nucleotides are as shown. The two Sp1 complexes are designated as A and C; the ets complex is designated B (arrows). Arrowheads mark the complexes of unresolved origin. P, position of the free probe.

Binding to the Sp1 Motif

Figure 3: Interactions with NHEK nuclear proteins and recombinant Sp1 protein with oligomers spanning the Sp1 recognition sequence. A, NHEK nuclear extract (lane 1) was combined with a probe spanning the sequences between -105 and -70 (lane 0). Lane 2 represents the competion of the NHEK binding with competitors in a 100-fold molar excess of the unlabeled probe. Lanes 3 and 4 show the competition of NHEK binding with the mutant and deletion variants shown below. Lane 5 is competition with consensus Sp1 oligonucleotide. B, same as A, but the binding reactions were done with recombinant Sp1 protein. The arrows denote the Sp1-specific complexes A and C. The letters in bold are the nucleotides of the Sp1 motif (boxed) that were mutated; dots represent unchanged nucleotides; P, position of the free probe.

Figure 4: Interactions with NHEK nuclear proteins with oligomers spanning the ets-like recoginition sequences. Binding of probe -134 to -102 (lane 0) to the NHEK nuclear extract in the presence of a 100-fold molar excess of poly(dI-dC) (lane 1) and or in the presence of a 100-fold molar excess of the corresponding competitor oligomers (lanes 2-13). The arrow denotes the ets-specific complex B. The letters in bold are the nucleotides in or adjacent to the ets motif (boxed) that were mutated; dots represent unchanged nucleotides; the arrowheads mark the complexes of unknown origin; P, position of the free probe.

Thus, whereas the Sp1 motif interacted with purified recombinant Sp1 protein to form the faster migrating complex C only, the interaction with the multicomponent NHEK extract resulted in the formation of both complex C and the slower migrating complex A. Moreover, when a probe carrying only the Sp1 motif was used, complexes A and C were of equal intensity (Fig. 3A), but when a longer probe carrying both the Sp1 and ets-like motifs was used, the intensity of complex C was always weaker (Fig. 2). Taken together these data suggest that complex C was not due to protein degradation of complex A, but rather, results from interactions of the Sp1 motif with Sp1 transcription factor alone. Complex A is likely to be due to a multicomponent interaction involving both Sp1, ets and/or other as yet unidentified proteins.

Binding to the ets Motif

Functional Analyses of the Proximal Promoter in Vivo

Figure 5: Functional analyses of the TGM3 proximal promoter region by transient CAT assays in NHEK cells. A, DNA sequence of the human TGM3 promoter region extending from -130 to -91. The ets-like and the Sp1-like recognition motifs are boxed. A-D represent the mutant variants used in the transient CAT assays. The letters in bold denote the mutated nucleotides. The dots represent the sequences which were not mutated. B, transient CAT activities of the wild type and mutant variants of the sequences between -130 and +10. The presentation of the relative CAT activities are as described in the legend to Fig. 1.

The involvement of ets and Sp1 transcription factors in the control of TGM3 promoter activity was further confirmed in co-transfection experiments. Simultaneous introduction into NHEK cells of the TGM3 construct -126/+10 with the pECE expression vector containing mouse ets-2 cDNA resulted in a 100% increase in the level of CAT compared to the level obtained on co-transfection of the same TGM3 construct with the pECE expression vector alone (Fig. 6). Co-transfection of the TGM3 construct with an Sp1 expression vector did not alter the activity of CAT, presumably because of high endogenous levels of this transcription factor in keratinocytes(67) . However, a further 30% increase in expression was obtained when vectors encoding both the ets-2 and Sp1 factors were simultaneously co-transfected (Fig. 6), thereby supporting the notion that ets-2 and Sp1 factors cooperate to regulate the expression of the TGM3 gene.

Figure 6: Synergistic effect of both ets-2 and Sp1 factors on the activity of TGM3 construct -126/+10. CAT activities were measured in NHEK cells that were co-transfected with the TGM3 construct -126/+10 (1 µg) and 0.5 µg of the pECE vector containing either no insert (designated as 100%) or the mouse ets-2 cDNA; the pRSV-Sp1 vector containing human Sp1 cDNA (1.0 µg); simultaneously with both parental vectors pECE and pRSV (0.5 µg each), containing the respective SV40 and RSV-long terminal repeat regulatory sequences only; simultaneously with the corresponding expression vectors for ets-2 and Sp1 cDNAs (0.5 µg each). The data are the averages of three or more independent experiments.

TGM3 Sequences between -126 and -91 Constitute a Powerful Promoter/Enhancer

Figure 7: The proximal promoter element located between -126 and -91 of the TGM3 gene confers high activity to a heterologous promoter in NHEK cells. The TGM3 sequences between -126 and -91 were cloned into a CAT reporter vector (ptk-TGM3) based on the ptk-Promoter (as described under ``Materials and Methods''). The data are expressed as percentages of the activity of the ptk-Promoter and are the averages of three independent experiments.

DISCUSSION

Our study demonstrates that the correct transcription of the human TGM3 gene, at least in vitro, depends on the simultaneous effect of elements residing in both the proximal promoter and in regions distal from it. The distal elements which are not present within the region extending 3.05 kb upstream of the transcription initiation site are required for late epidermal differentiation-specific transcription. The proximal promoter of the TGM3 gene, however, contains the information sufficient to direct high levels of expression in stratified squamous epithelial cells in culture. We have mapped this region to sequences between -126 and -91, which encompass Sp1 and ets-like binding sites. The overall activity of the proximal promoter region results from the cooperative interactions between these positively acting motifs.

Keratinocytes contain relatively high levels of Sp1(67) . This transcription factor is important for the regulation of a variety of other epidermally-expressed genes(68, 69, 70) . On the other hand, the TGM3 gene is the first epidermally-expressed gene in which the ets transcription factors have been shown to be important. These transcription factors have been implicated in the regulation of gene expression during a variety of biological processes, including growth control, developmental or transformation programs, and usually function as components of larger transcription complexes(65) . Indeed, our transient transfection data ( Fig. 5and Fig. 6) demonstrated cooperative interactions between ets and Sp1 transcription factors, as has been previously reported in other gene systems(70, 71, 72, 73) . However, in the TGM3 gene proximal promoter region, the interactions clearly involved not only Sp1, but also additional proteins in the NHEK nuclear extracts (Fig. 3). Thus the cooperative relationship between the ets and Sp1 factors in the activity of the TGM3 proximal promoter is modulated by interactions with additional so far unidentified nuclear proteins.

Our present data on the expression of the TGM3 gene in keratinocytes represent the first detailed study on the regulatory elements involved in the transcription of either of the three transglutaminase genes expressed in epithelia. To date in the TGM1 gene, a 0.82-kb fragment could confer epithelial specific expression only in TPA-treated cells (57) . In the case of the TGM2 gene, an initial study showed that 1.6 kb of flanking DNA sequences contain low core promoter activity in both epithelial and non-epithelial cell lines, at a level comparable to our construct -91/+10 (Fig. 1; Table 1). Deletion analyses indicated that putative Sp1 binding motifs may be responsible in the TGM2 gene(56) . In this regard we have examined the upstream sequences of the three transglutaminase genes for common regulatory elements. All three contain consensus Sp1 recognition motifs near the transcription start site. Interestingly, the TGM1 gene ()also possesses an ets-like motif at position -190. Thus it will be interesting to determine whether the Sp1 and ets motifs can also confer high levels of epithelial specific expression to the TGM1 gene. Furthermore, it raises the possibility that members of the ets family of transcription factors may be important in the regulation of late differentiation genes in the epidermis.

To date, only two of the several genes involved in the latest stages of epidermal differentiation have been studied, the TGM3 gene, as reported here, and the loricrin gene. About 2.5 kb of upstream sequences are required for epithelial specific expression of the loricrin gene(74) , and 10 kb are required for correct temporal expression(74, 75) . The proximal promoter region which confers high levels of epithelial specific expression of the TGM3 gene in cultured keratinocytes is located within a narrow window of -126 to -91 base pairs of the transcription start site. This promoter should now be very useful for further studies on the expression of this and other genes in keratinocytes. Furthermore, it may aid in devising strategies to manage lamellar ichthyosis (46, 47, 48) and other related recessive genodermatoses involving mutations in the transglutaminase genes.

FOOTNOTES

*

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.

§

Present address: Dept. of Dermatology, Chungnam National University Hospital, Daejeon, Republic of Korea.

¶

Present address: Dept. of Dermatology, Samsung Medical Center, Seoul 135-230, Republic of Korea.

**

To whom all correspondence should be addressed. Tel.: 301-496-1578; Fax: 301-402-2886; :pemast{at}helix.nih.gov.

()

The abbreviations used are: TGase 3, transglutaminase 3; -gal, -galactosidase; CAT, chloramphenicol acetyltransferase; NHEK, normal human epidermal keratinocytes; TGase, transglutaminase; TGM3, transglutaminase 3 gene; tk, thymidine kinase; kb, kilobase (s); RSV, Rous sarcoma virus; TPA, 12-O-tetradecanoylphorbol-13acetate; DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammoniummethylsulfate.

()

N. G. Markova, unpublished observations.

ACKNOWLEDGEMENTS

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