The eta isoform of protein kinase C mediates transcriptional activation of the human transglutaminase 1 gene.

Transglutaminase 1 (TGase 1) is expressed during the terminal differentiation of keratinized squamous epithelium to form cornified cell envelope in differentiated keratinocytes by the epsilon-(gamma-glutamyl) cross-linking reaction. The gene for human TGase 1 is responsible for autosomal recessive lamellar ichthyosis, a severe hereditary keratinizing disorder of the skin. We examined the transcriptional activity of the gene in FRSK, rat keratinocytic cells, transfected with the luciferase reporter gene under control of the 5' upstream region of human TGase 1 gene. Transfection of the reporter gene with an expression vector for the eta isoform of novel protein kinase C (nPKCeta), as well as exposure to 12-0-tetradecanoylphorbol-13-acetate, markedly increased the luciferase activity in FRSK, but not in HT-1080 fibrosarcoma cells, although exogenous nPKCeta was expressed in both. The induction was suppressed by deleting the TGase 1 upstream sequence from -95 to -67 and by deleting the kinase domain from exogenous nPKCeta. In comparison with other PKC isoforms, nPKCeta most effectively induced the luciferase activity. We suggest that nPKCeta, an epithelium-specific isoform of PKC, mediates the activation of the TGase 1 transcription.

Transglutaminase 1 (TGase 1) is expressed during the terminal differentiation of keratinized squamous epithelium to form cornified cell envelope in differentiated keratinocytes by the ⑀-(␥-glutamyl) cross-linking reaction. The gene for human TGase 1 is responsible for autosomal recessive lamellar ichthyosis, a severe hereditary keratinizing disorder of the skin. We examined the transcriptional activity of the gene in FRSK, rat keratinocytic cells, transfected with the luciferase reporter gene under control of the 5 upstream region of human TGase 1 gene. Transfection of the reporter gene with an expression vector for the isoform of novel protein kinase C (nPKC), as well as exposure to 12-O-tetradecanoylphorbol-13-acetate, markedly increased the luciferase activity in FRSK, but not in HT-1080 fibrosarcoma cells, although exogenous nPKC was expressed in both. The induction was suppressed by deleting the TGase 1 upstream sequence from ؊95 to ؊67 and by deleting the kinase domain from exogenous nPKC. In comparison with other PKC isoforms, nPKC most effectively induced the luciferase activity. We suggest that nPKC, an epithelium-specific isoform of PKC, mediates the activation of the TGase 1 transcription.
Terminal differentiation is a highly organized biological process that involves the programmed death of cells (1). The epidermis covering the most outer layer of the skin is maintained by a dynamic equilibrium of renewal with terminal differentiation of keratinocytes, the major cell lineage of the epidermis. This process, keratinization, proceeds with the ordered expression of genes in keratinocytes and their morphological changes. Transglutaminase 1 (TGase 1), 1 a member of the transglutaminase family (EC 2.3.2.13), is expressed during keratinization (2)(3)(4). The enzyme catalyzes the ⑀-(␥-glutamyl) lysine cross-linking reaction of the proteins, such as involucrin (5), cystatin-␣ (6), loricrin, small proline-rich proteins, and elafin (7), and forms the cornified cell envelope on the inner surface of the differentiated keratinocytes (4). This insoluble structure maintains the internal milieu and protects the body against the chemical, mechanical, and biological invasion. The importance of this enzyme in the morphogenesis and barrier function of the skin has been shown also by discovery of its mutation in autosomal recessive lamellar ichthyosis (8 -10), which is a hereditary keratinization disorder characterized by collodion baby at birth and later, severe ichthyosis with large brownish plate-like desquamations and epidermal hyperkeratosis, ectropion, eclabium, and scarring alopecia.
The gene for human TGase 1 is located on 14q11.2 (11,12) and encodes a 92-kDa protein consisting of 816 amino acid residues (13)(14)(15). In the epidermis of the skin, TGase 1 gene is expressed mainly in the subcorneal granular layers (16 -18). The enzyme activity of transglutaminase is enhanced by treating primary mouse epidermal cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C (19). In cultured epidermal keratinocytes and tracheal epithelial cells, the level of TGase 1 mRNA is increased also by TPA (20,21) as well as Ca 2ϩ (22)(23)(24), ganglioside GQ 1b (24), and interferon-␥ (25). Besides the effect of TPA, the critical function of PKC in the regulation of TGase 1 gene expression has been suggested by studies using several PKC inhibitors (20,26). However, there is no direct evidence indicating that PKC mediates the signal for the transcriptional activation of TGase 1.
PKC was initially characterized as a serine/threonine-type protein kinase, the activity of which is dependent on Ca 2ϩ , phospholipids, and diacylglycerol (27)(28)(29). Subsequently, various isoforms of the enzyme have been identified (30). PKC is now classified into three major subgroups, Ca 2ϩ -, phospholipid-, and diacylglycerol-dependent classical PKCs (cPKC) (␣, ␤I, ␤II, and ␥), Ca 2ϩ -independent novel PKCs (nPKC) (␦, ⑀, , and ) and Ca 2ϩ -and diacylglycerol-independent atypical PKC (aPKC) ( and /). In addition, the isoform, a membranebound PKC, has been identified (31). They differ in terms of tissue distribution and are involved in a variety of specialized functions of the tissues. One nPKC, called nPKC (PKC-L), is epithelium-specific (32,33). Although there are also other PKC isoforms, such as cPKC␣ and nPKC␦, in the epidermis of the skin (34), the localization of nPKC in the subcorneal granular layer is quite similar to that of TGase 1 mRNA (16,17), suggesting that it mediates the activation signal for TGase 1 gene during the terminal differentiation of epidermal keratinocytes. Understanding the unique signal transduction for TGase 1 gene regulation should provide new insight into the molecular mechanism involved in the complex biological process of keratinization. Therefore, we analyzed the role of nPKC in the transcriptional activation of TGase 1 gene expression.

EXPERIMENTAL PROCEDURES
Materials-FRSK cells (35,36) and HT-1080 fibrosarcoma cells (37) were supplied by the Japanese Cancer Research Resources Bank (Foundation for Promotion of Cancer Research, Tokyo, Japan). The luciferase reporter plasmids PGV-B and PGV-C were purchased from Toyo Ink Manufacturing Co., Ltd. and 12-O-tetradecanoylphorbol-13-acetate was obtained from LC Service Corp. Oligonucleotides were synthesized using a DNA synthesizer (model 392; Applied Biosystems, Inc.). All other reagents and chemicals were purchased from commercial sources and were either of reagent grade or the highest purity available.
Cell Culture-FRSK cells were cultured in Eagle's minimum essential medium supplemented with 10% fetal calf serum, and HT-1080 cells were cultured in the same medium containing nonessential amino acids (ICN Biochemicals Inc.) at 37°C under 5% CO 2 in air. Cells were subcultured every 3-4 days until they reached 80% confluence.
Construction of Reporter Plasmids-Deletion fragments of the 5Ј upstream region of human TGase 1 gene from Ϫ819 to Ϫ49 were generated by the polymerase chain reaction (PCR) using a plasmid pd1L as the template (38). These fragments were amplified using UlTma DNA polymerase (Perkin-Elmer) and synthetic oligonucleotide primers, according to the manufacturer's instruction. These PCR products and the synthesized oligonucleotides for Ϫ20 deletion were inserted into the SmaI site of PGV-B, a luciferase reporter plasmid containing neither enhancer nor promoter sequences. These reporter plasmids are summarized in Fig. 1. PGV-C is a constitutive expression plasmid for luciferase containing the SV40 promoter and enhancer.
Construction of SRDRD-The kinase domain of mouse nPKC is encoded by a 865-bp sequence from nucleotides 1041-1905 of the cDNA (33). SRD, an expression plasmid containing the complete coding sequence for mouse nPKC (33), was digested with EcoRI and SalI. The 1.0-kb fragment encoding the E region and the regulatory domain of the enzyme was inserted into the EcoRI site of pcDL-SR␣296, the backbone of SRD (39).
Transient Expression Assay-Cells were seeded at a density of 6 ϫ 10 4 cells/2 ml of medium in 35-mm dishes and incubated at 37°C under 5% CO 2 in air. Two days later, the DNA was transfected into the cells by lipofection using N- [1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) (Boehringer Mannheim), according to the manufacturer's instructions. Transfected DNA consisted of 1 g of reporter plasmid with or without expression plasmids, 0.1 g of pCMV␤ (40) as an internal control for variation in transfection efficiencies, and the appropriate amount of pGEM3Z (Promega) to standardize the total amount of DNA. After an incubation at 37°C for 16 h, the medium was changed and 20 h later the cells were exposed or not to 1 nM TPA for 4 h, then harvested for luciferase and ␤-galactosidase assays or processed for immunoblotting. Luciferase activity was assayed with a PicaGene kit (PGK-L500, Toyo Ink Manufacturing Co., Ltd.) using a Monolight 2010 luminometer (Analytical Luminescence Laboratory). We deter-mined ␤-galactosidase activity using a ␤-galactosidase enzyme assay system (Promega) and a microplate reader (model 450, Bio-Rad). The experiment was repeated at least three times, and representative results are shown in the figures. The luciferase activities from duplicate experiments were normalized relative to ␤-galactosidase activities and protein concentrations.
Immunoblotting Analysis-Cells were immersed in 10% trichloroacetic acid at 4°C for 15 min, then scraped off the dishes. After centrifugation at 10,000 ϫ g for 5 min, the trichloroacetic acid was removed and the pellet was homogenized in 80 l of Urea-TX (9 M urea, 2% Triton X-100, 5% 2-mercaptoethanol) by sonication on ice. To this, 20 l of 10% lithium dodecyl sulfate and 1 l of 2 M Tris were added, then the proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (41). The separated proteins were electrically transferred onto a Clearblot-P membrane (Atto Corp.). The blot was probed with anti-PKC␣ antibody (Life Technologies, Inc.), anti-nPKC antibody (Santa Cruz Biotechnology, Inc.), and anti-nPKC␦ antibody (42). The protein was detected using second anti-rabbit IgG antibody conjugated with alkaline phosphatase (Bio-Rad).

RESULTS
The Transcriptional Activity of the 5Ј-Upstream Region of Human TGase 1 Gene-FRSK is a keratinocytic cell line de- The TGase 1-luciferase reporter genes were transfected into FRSK, cultured rat keratinocytes. After a 2-day incubation, the cells were exposed or not to 1 nM TPA for 4 h, and then the luciferase activity was determined, as described under "Experimental Procedures." B, the effect nPKC on the transcriptional activity of human TGase 1. SRD, an expression plasmid for nPKC, was cotransfected with the reporter genes into FRSK cells; 2 days later, the luciferase activity was determined. Data represent the means of duplicate experiments normalized to ␤-galactosidase activity. PGV-B and PGV-C are negative and positive control reporter plasmids, respectively.
FIG. 1. The TGase 1-luciferase reporter genes. Deletions of the human TGase 1 5Ј flanking region from Ϫ819 to Ϫ49 were generated by PCR and inserted into the SmaI site of PGV-B, a basic luciferase reporter vector. PGV-C is a constitutive expression vector for luciferase driven by SV40 promoter and enhancer. Arrows indicate PCR primers used to synthesize the deletion mutants. Upper primers are as follows: pd-819, aattccaggttggagaaaagg; pd-95, cccatttcccgcccagaggcc; pd-66, tctctccgccccctacagcag; pd-49, gcagtttggcccctccctccc. The lower primer is tcacttaccaggtctgtccct. This primer was also used to prepare the deletion fragment pd-20, by annealing it with its sense oligonucleotide. E, EcoRI; P, PvuII; B, BamHI; H, HindIII; Luc, luciferase gene; SV40 box, SV40 enhancer; P box, SV40 promoter. rived from fetal rat skin (35,36). FRSK cells, like HaCaT cells derived from a human epidermoid carcinoma, are grown in medium with a normal Ca 2ϩ concentration. We have studied the transcriptional regulation of human TGase 1 gene by means of electroporation (38). In this study, we adopted lipofection using DOTAP, because the transfection efficiency of this procedure was 10-fold higher than that of electroporation. There was practically no difference in the transcriptional activities between the 2.5-kb and 819-bp upstream regions of TGase 1 gene (38). Therefore, we examined the activity of the region up to Ϫ819 of the gene. When the luciferase reporter plasmid pd-819 containing the 819-bp upstream sequence of human TGase 1 gene was transfected into FRSK cells by lipofection, there was substantial basal luciferase activity ( Fig.  2A). The activity was potentiated by 1 nM TPA, which induces the expression of TGase 1 mRNA in keratinocytes (20). To assess the sequence required for the transcriptional activation of the gene, we prepared deletion mutants of its 5Ј upstream region from Ϫ819 to Ϫ20 and luciferase reporter plasmids containing these sequences were transfected into FRSK cells. As shown in Fig. 2A, the luciferase activity remained even when the sequence from Ϫ819 to Ϫ96 was deleted from the upstream region. However, the major loss of TPA-stimulated luciferase activity was seen when the sequence between Ϫ95 and Ϫ67 was deleted. A deletion from Ϫ66 to Ϫ50 substantially reduced the basal luciferase activity. These results suggest that the induction of TGase 1 gene expression by TPA is regulated at the transcriptional level and that this 29-bp 5Ј upstream sequence is required for the signaling.
The Activation of Human TGase 1 Transcription by nPKC-Of the PKC family, the localization of nPKC is closely related to that of TGase 1 mRNA in human epidermis (16,17,34,43), implying that it is involved in the induction of TGase 1 transcription. To investigate this notion, we co-transfected SRD (33), an expression plasmid for mouse nPKC, into FRSK cells with the same set of reporter constructs used for the experiment described above. As shown in Fig. 2B, the co-transfection of SRD with pd-819 increased the luciferase activity and the induction was comparable to that by TPA. The effect of the co-transfection was abolished by deleting the 5Јupstream sequence from Ϫ95 to Ϫ67, which was the same region required for the effect of TPA. These findings indicated that nPKC mediates the activation of TGase 1 transcription.
The Effect of nPKC on the TGase 1 Transcription in Different Cell Types-To study the dose-responsive effect of nPKC, various amounts of SRD were transfected into FRSK cells with the reporter plasmid pd-95, which contains the minimum proximal upstream sequence responsive to both TPA and nPKC. As shown in Fig. 3A, the increase in the luciferase activity was maximal at 12.5 and up to 500 fmol of SRD. The level of the induction by SRD was similar to that elicited by co-transfection with 500 fmol of pL27, an expression plasmid for human nPKC (44).
In contrast to FRSK cells, TPA fails to activate TGase 1 transcription in HT-1080, a human fibrosarcoma cell line, even when a reporter plasmid containing Ϫ819 upstream sequences is transfected (38). When pd-95 was co-transfected with SRD from 2.5 to 25 fmol into HT-1080 cells, the induction of luciferase activity remained at low levels (Fig. 3B), indicating that the machinery that transduces the signal elicited by nPKC is defective in HT-1080 cells.
Deletion of the Kinase Domain of nPKC Abolishes Its Effect on Human TGase 1 Gene-The enzyme nPKC is composed of a regulatory and a kinase domain. Unlike classical PKCs, the regulatory domain of nPKC has no Ca 2ϩ binding subdomain, but its kinase domain is homologous to those of other PKCs. To study the role of the kinase domain of nPKC in the activation of TGase 1 transcription, we constructed the plasmid SRDRD from SRD by deleting the cDNA sequence encoding this domain and co-transfected this plasmid with pd-95 into FRSK cells. SRDRD did not induce luciferase activity over the basal level (Fig. 4). TPA caused no additional increase in the luciferase activity in FRSK cells transfected with SRDRD as well as SRD. Thus, nPKC is essential for TGase 1 gene expression and possibly the kinase domain of the enzyme is involved in the signaling for the transcription.
Expression of PKC Isoforms by Transfection with Their Expression Plasmids-The effect of nPKC expression on human TGase 1 transcription was almost a plateau, when over 12.5 fmol of SRD were transfected into FRSK cells (Fig. 3A). This might be due to the limitation on the expression level of exogenous nPKC in FRSK cells. A similar mechanism might lead to low levels of activation of TGase 1 transcription in HT-1080 cells transfected with SRD. These possibilities were ruled out by immunoblotting extracts from the cells transfected with SRD using an anti-nPKC antibody, which was raised against a peptide corresponding to a 15-amino acid sequence within the C terminus of the enzyme. An 82-kDa band corresponding to exogenous nPKC was detected in both FRSK (Fig. 5A) and HT-1080 cells (Fig. 5B). The level of nPKC expression was increased depending on the amount of transfected SRD, at least, in the range up to 250 fmol. We examined the expression of the PKC isoforms PKC␣ and PKC␦ in FRSK cells transfected or not with each expression plasmid (33,39,42). As shown in Fig. 5 (C and D), the increases in the expression of these PKC isoforms were also dose-dependent. In the range up to 250 fmol of these effector plasmids, there was no limitation on the expression of the exogenous proteins.
Comparison of the Effect of nPKC with Other PKC Isoforms on the Human TGase 1 Transcription-In epidermal keratinocytes, at least cPKC␣, nPKC␦, and nPKC are expressed (34,45). cPKC␤II is found in Langerhans cells of the skin as well as other organs (46), and cPKC␥ is predominantly expressed in the brain. aPKC isoform is expressed in a variety of tissues (47). In addition to nPKC, we examined the effects of other PKC isoforms on TGase 1 transcription in FRSK cells. The expression plasmids for cPKC␣, cPKC␤II, cPKC␥, nPKC␦, nPKC, or aPKC (33,39,42) were co-transfected with pd-95, and the luciferase activity was compared. As shown in Fig. 6F, nPKC most efficiently induced luciferase activity. Next to nPKC, nPKC␦ was also effective (Fig. 6E). However, cPKC␣, cPKC␤II, cPKC␥, and aPKC hardly induced the luciferase activity (Fig. 6, A-D and G). Thus, among the PKC isoforms examined, nPKC was the most efficient activator of human TGase 1 transcription.

DISCUSSION
Several reports have provided evidence that PKC regulates TGase 1 gene expression. We found that TPA, an activator of PKC, increases TGase 1 mRNA in cultured normal human epidermal keratinocytes and this effect is suppressed by the PKC inhibitors H-7 and staurosporine (20). Dlugosz and Yuspa (26) have also shown that the effect of TPA is blocked by the PKC inhibitors, GF109203X and bryostatin 1, in cultured primary mouse epidermal keratinocytes. They also postulated that TGase 1 mRNA expression is regulated at the transcriptional level, from the results of a nuclear run-on assay. The transcriptional regulation by TPA has been confirmed in rabbit and human TGase 1 genes by means of a transient expression assay using the 5Ј upstream region of the genes (21,38). In this study, we show further direct evidence that nPKC, an isoform of PKC, regulates the transcriptional activation of the TGase 1 gene. This mechanism may also be functional in the epidermis of the skin, because the localization of nPKC is closely associated with that of TGase 1 mRNA in normal and psoriatic epidermis (16,17,34,43).
Deletion of the 5Ј upstream region of the human TGase 1 gene indicates that the sequence from Ϫ95 to Ϫ67 includes critical elements in response to both nPKC and TPA. The overexpression of nPKC with TPA induces no additional potentiation of its transcription in FRSK and nPKC without the kinase domain cannot increase the transcriptional activity. Thus, it is likely that TPA activates nPKC and the signal generated by phosphorylation is concentrated on the elements of the upstream region. Saunders et al. (21) have demonstrated the promoter activity of the proximal upstream region of rabbit TGase 1 gene. Its activation requires the sequence from Ϫ95 to Ϫ50 of the gene, the position of which is similar to that of human TGase 1 gene (12). However, the homology of their sequences was not so high, suggesting a difference in TGase 1 gene regulation between the species.
As shown by immunoblotting, exogenous nPKC is well expressed in FRSK as well as HT-1080 fibroblastic cells. An increase in the expression levels of nPKC by exogenous introduction of an nPKC expression plasmid activates the transcription of TGase 1 in FRSK cells, but to a very limited extent in HT-1080 cells. Therefore, the signaling for TGase 1 gene expression is probably mediated by nPKC, but its presence is not necessarily sufficient to induce the transcriptional activation of the gene.
In terms of ability to induce TGase 1 transcription, nPKC was the most potent of all the PKC isoforms tested, cPKC␣, cPKC␤II, cPKC␥, nPKC␦, and aPKC. This prominent efficiency of nPKC seems to represent its selective importance in epidermal differentiation. It is localized in rough endoplasmic reticulum (48), and, unlike cPKCs, it cannot be translocated to the plasma membrane by activation (45). This unique feature of nPKC may be related to the induction of TGase 1 transcription. We also find that nPKC␦ can induce TGase 1 transcription to some extent, suggesting possible contribution of the gene expression, although the PKC isoform is expressed in various organs including epidermis (45,47).
There is neither a conventional nor other TPA-responsive elements in the 5Ј upstream sequence from Ϫ95 to Ϫ67, which is critical to TGase 1 transcription (12). We pointed out two Sp1 motifs in the 5Ј proximal upstream region of human TGase 1 gene (12). Indeed, when a Sp1 expression plasmid was introduced into FRSK cells with a reporter plasmid pd-95, the luciferase activity increased (data not shown). Hence, we speculate that Sp1 or Sp1-related factors are involved in the transcriptional activation of TGase 1 gene. Further studies on the characterization of the signaling system via nPKC will provide new insights in the control of TGase 1 gene expression, which will lead to the elucidation of the molecular mechanisms involved in epidermal terminal differentiation and the molecular pathogenesis of keratinizing disorders, including lamellar ichthyosis.