Introduction

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In vivo squamous-cell metaplasia in the airway represents a multistep change in surface epithelium from a predominately mucociliary cell layer to a layer of cells with squamous appearance (1, 2), usually in response to injury/repair and vitamin A deficiency. Normally, there is an initial change in stratification of epithelial cell morphology at or near the injury area, followed by an enhanced cell proliferation and expression of various squamous-cell markers and, finally, the expression of terminal squamous-cell differentiation, such as keratinization and cornification, as a scar in the region (1). The nature of such a transepithelial change in vivo in airway epithelium is not clear. However, it has been suggested as one of the preneoplastic lesions associated with bronchogenic cancer development (4, 5).

In vitro, the terminal squamous differentiation of conducting airway epithelial cells is also a multistep process featuring the expression of many squamous-cell markers such as involucrin, loricrin, SPRR1, etc. (6, 7), and the formation of cross-linked or cornified envelopes (8). Phorbol ester potently induces terminal squamous differentiation in both keratinocytes and airway epithelium (9, 10). We found that although both cell cornification and SPRR1 gene expression are enhanced by phorbol ester in cultured human bronchial epithelial cells (11, 12), the signaling pathways leading to these enhancements are not completely understood.

The main intracellular receptor for phorbol ester is protein kinase (PK)C (13). The activation of PKC upon tetradecanoyl phorbol acetate (TPA) treatment has been observed in many cell types (14). Experiments with keratinocyte cultures strongly suggest that the induction of squamous differentiation by TPA is mediated through a direct activation of PKC (10). Our previous experiments with a PKC inhibitor, sphingosin, and the receptor downregulation approaches have demonstrated the inhibition of TPA-dependent stimulation of SPRR1 gene expression (11). It is possible that both cell cornification event and SPRR1 gene expression enhancement by phorbol-13-myristate-12-acetate (PMA) are mediated by PKC-dependent signaling pathways in airway epithelium.

Mitogen-activated protein kinases (MAPKs) are common intermediates in intracellular signaling cascades involved in diverse cellular functions, including growth and differentiation (15, 16). The MAPK family includes p42/ 44MAPK (also referred to as extracellular regulated kinase [ERK] 2 and 1, respectively) (17), the c-Jun N-terminal kinase (also referred to as stress-activated kinase) (18), and p38 (19). The activation of p42/44MAPK requires the dual phosphorylation of Thr and Tyr residues by the activated kinase, MAPK kinase (MEK1 and MEK2) (20, 21). Raf-1 and B-Raf are serine/threonine-protein kinases that selectively phosphorylate and activate MEK1 and MEK2 (22). The activation of MAPK by phorbol ester has been established in many cell types (26), and occurs through Ras-Raf-MEK-ERK or Raf-MEK-ERK pathways, depending on the type of PKC isoenzymes that are activated (30). Because we observed the elevation of both cell cornification and SPRR1 gene expression by phorbol ester in airway epithelium, we wondered whether these elevations were mediated by MAPK.

In addition to PKC activation, phorbol ester is known to bind to other cellular components, such as Ras-activating guanine nucleotide exchange factor and Rac-guanosine triphosphatase-activating protein (26, 27). This raises the question of whether phorbol ester-induced cell cornification and SPRR1 gene expression in airway epithelium are mediated soley by PKC. To clarify this issue, we used a more specific PKC activator and inhibitor in this study and demonstrated that the enhancement of both cell cornification and SPRR1 gene expression by phorbol ester is mediated by a PKC-dependent pathway. We have further shown that the phorbol ester-induced SPRR1 gene expression occurs through an MAPK pathway, whereas cell cornification takes place through an MAPK-independent pathway.

	Materials and Methods

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Cell Culture

Human tracheobronchial tissues were obtained from the Medical Center of the University of California, Davis (Davis, CA), and Sacramento Organ Donation Foundation, Inc. (Sacramento, CA). All procedures involved in tissue procurement were approved by the University of California, Davis, Human Subjects Review Committee. Epithelial cell isolation and culture were carried out as described previously, with some modifications (31, 32). Primary tracheobronchial epithelial (TBE) cells were grown in F12/ Dulbecco's modified Eagle's medium (DMEM) (1:1) supplemented with six hormonal factors and bovine serum albumin (0.5 mg/ml) until confluence occurred. Preparation of hormonal supplements has been described elsewhere (28), and the doses used in the culture medium were as follows: insulin (5 µg/ml), transferrin (5 µg/ml), epidermal growth factor (10 ng/ml), cholera toxin (10 ng/ml), dexamethasone (0.1 µM), and bovine hypothalamus extract (15 µg/ml). Under this modified culture medium, primary cells grew rapidly in culture, and confluence was achieved within 7 to 10 d with a seeding density of 1 to 2 × 10⁴ cells/cm² culture area. The epithelial nature and mucociliary differentiation potential of cultured cells were confirmed by antikeratin antibody (AE-1 and AE-3) staining and by the tracheal graft repopulation methods, respectively (data not shown). Confluent primary cultures were then trypsinized and plated on tissue culture dishes with a cell density of 5 × 10⁴ cells/cm² in a serum-free hormone-supplemented keratinocyte basal medium (KBM) (Bio Whittaker, San Diego, CA) at the 0.09-mM calcium level. The hormonal supplements are those six factors used in the primary cultures. Passage 1 cultures were maintained in this low-calcium medium for 6 d before the shift to different experimental conditions. The induction of cell cornification was carried out in the F12/DMEM medium by the addition of PMA (5 ng/ml).

For all the inhibitor experiments, the cells were preincubated with inhibitors and activators for 1 h before PMA treatment. These inhibitors, bisindolylmaleimide (BIM) I and PD98059, and the activator, indolactam V, were obtained from Calbiochem (San Diego, CA) and they were freshly prepared in dimethyl sulfoxide (DMSO) and used immediately upon their arrival. The amounts of these chemicals used were 0.1 to 10 µM, 0.01 to 1.0 µM, and 50 to 100 µM for indolactam V, BIM I, and PD98059, respectively. The control cultures were treated with the same level of DMSO, usually less than 0.1%.

Cross-Linked Envelope Assay

Cross-linked envelopes were quantified by the Jetten and Smits method with some modifications (33). Briefly, after trypsinization, cells were resuspended in 1 ml phosphate-buffered saline (PBS) and total cell number was counted under a microscope with a hemocytometer. A portion of the cell suspension was centrifuged for 10 min at 1,000 × g and then treated with 100 µl lysis solution containing 2% sodium dodecyl sulfate (SDS) and 2% -mercaptoethanol for 5 min. Undissolved envelopes observed under a hemocytometer were quantified as crosslinked. The percentage of cross-linked envelopes was determined by dividing the number of undissolved cells by total number of cells present. Each counting involved an average of four to six different areas of each hemocytometer slide. Each cell suspension was counted three times with different hemocytometer slides, and averaged. Triplicate dishes were used for each cell counting, and the data are presented as averages with a standard deviation (SD). Each data point was independently verified by at least two different passage 1 cultures generated from two different primary tissues.

RNA Isolation and Northern Blot Hybridization

Total RNA was isolated from culture cells by the single-step acid guanidinium thiocyanate-phenol-chloroform extraction method, as previously described (11, 34). For Northern blot, equal amounts of total RNA (20 µg/lane) were subjected to electrophoresis on a 1.2% agarose gel in the presence of 2.2 mM formaldehyde and transblotted onto Nytran membranes. The RNA was cross-linked to membrane by UV Stratalinker 2400 (Stratagene, La Jolla, CA). The membranes were prehybridized at 68°C in a solution containing 6× saline sodium citrate (SSC), 10 mM ethylenediaminetetraacetic acid, 5× Denhardt's solution, 0.5% SDS, and 100 µg/ml of sheared salmon sperm DNA. The complementary DNA insert of monkey SPRR1 and 18S were [³²]P-labeled by the multiprimer DNA labeling system. The hybridization was performed overnight at 68°C. The membrane filters were washed three times for 30 min each at 68°C with 2× SSC/0.1%SDS, 1× SSC/0.1% SDS, and 0.1× SSC/0.1% SDS, respectively. The filters were then dried and subjected to autoradiography using Kodak XAR-5 film. The relative abundance of SPRR1 messenger RNA (mRNA) in cells was normalized with 18S rivosomal RNA (rRNA) band.

Western Blot Analysis of ERK Activation

Passage 1 human TBE cultures were treated with PD98059 (MAPK inhibitor) 1 h before the PMA treatment (5 ng/ml). At 5, 15, and 30 min after the PMA treatment, cells were lysed in a lysis buffer containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 30 µl/ml aprotinin, and 1 mM sodium orthovanadate (Na₃VO₄) in PBS. A total of 40 µg of cell lysate protein from each time point was subjected to a 10% SDS-polyacrylamide gel electrophoresis (PAGE). After electrophoresis, proteins were electroblotted onto Immobilon-P membrane (Millipore, Bedford, MA) and incubated with anti p-ERK monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Western blot analysis was carried out as described elsewhere (35).

Statistical Analysis

Data are expressed as means ± SD, on the basis of at least triplicate dishes. Each data point was confirmed at least twice from two different primary tissue sources. Group differences were analyzed by analysis of variance. When P < 0.05, the difference was considered significant.

	Results

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Time-Course Effects of PMA on the Induction of Cell Cornification and SPRR1 Message

Under the described culture conditions, the cornified cell population was minimalless than 10% of cornified cell population was observed throughout the passage 1 culture (Figure 1A). When the confluent culture was treated with PMA at the 5 ng/ml level, it induced a significant change in cell morphology (Figure 2B) and enhanced cell cornification. Within 24 h of treatment, the cross-linked cell population was at the 30% level throughout the culture. A similar enhancement on the SPRR1 message level by PMA was observed. The time-course study demonstrated that this increase occurs within 8 h and decreases at 48 h after the PMA treatment (Figure 1B). A 3-fold level of enhancement occurred when normalizing with 18S rRNA (Figure 1C).

View larger version (41K): [in this window] [in a new window]   View larger version (15K): [in this window] [in a new window]   Figure 1.   Effects of PMA on cell cornification and SPRR1 gene expression in passage 1 human TBE cells. Human TBE cells were propagated as described in MATERIALS AND METHODS. PMA was added at 5 ng/ml and cultures were harvested at various times as indicated for (A) cell cornification and (B and C) RNA Northern blot hybridization for SPRR1 gene. The cell cornification study was carried out as described in MATERIALS AND METHODS and expressed as a percent of cell cornified. Quantitative data for SPRR1 message level normalized with the 18S rRNA data are present in C. Experiments were repeated in three passage 1 cultures from different human airway tissues with similar results. The data represent one of these experiments.

View larger version (188K): [in this window] [in a new window]   Figure 2.   Cell morphology under phase contrast microscope. Passage 1 human TBE cells were maintained in culture, as described in MATERIALS AND METHODS. The near-confluent cultures were shifted to three different experimental conditions (B, PMA; C, PMA/BIM I [106 M]; and D, indolactam V [106 M]); or maintained in a control culture condition (A). The pictures were taken 24 h after the treatments.

Effects of PKC Inhibitor on PMA-Induced Cell Cornification

To clarify the question of whether enhancement by PMA is mediated through the activation of PKC, a PKC-specific inhibitor, BIM I, and a PKC-specific activator, indolactam V, were used to further establish the role of PKC in PMA-stimulated cell cornification and SPRR1 gene expression. Human passage 1 TBE cells were maintained in KBM medium with 0.09 mM of calcium and six factors until confluence and then treated with these chemicals. As shown in Figure 2, both PMA and indolactam V treatments caused similar morphologic change on cultured cells. These changes include a flattened cell morphology with a decrease in nucleus-to-cytosol ratio, which could be prevented by the PKC-specific inhibitor BIM I (Figure 2C). BIM I alone had no effect on cell morphology (data not shown).

In addition to the cell morphology effect, within 24 h there was an increase of cross-linked envelope formation in cultures treated with PKC-specific activators. Both PMA and indolactam V enhanced cell cornification from the 5 to the 35% basal level. Indolactam V at the 100-nM level was as effective as PMA in causing cell cornification (Figure 3). This increase of cell cornification was not related to the toxicity of these chemicals, inasmuch as a trypan blue dye exclusion assay revealed more than 98% viability in culture after the treatment. In contrast to the PKC activators, BIM I was able to reduce PMA-induced cell cornification. This prevention was dose-dependent (Figure 3), further suggesting a specific PKC-dependent signaling pathway involved in the induction of cell cornification. These results further support the notion that the enhancement of cell cornification by PMA is mediated by PKC.

View larger version (46K): [in this window] [in a new window]   Figure 3.   Effects of PKC activator and inhibitor on the cross-linked envelope formation in passage 1 human TBE cells. Passage 1 TBE cultures were carried out as described in Figure 1. Before PMA (5 ng/ml) treatment, cultures were pretreated with different concentrations of indolactam V and BIM I, as shown. Cross-linked envelopes were quantified 24 h after the PMA treatment. The data are presented as means ± SD from triplicate dishes. Duplicate experiments were performed in passage 1 TBE cells isolated from another human subject.

Effects of MAPK Inhibitor on PMA-Induced Cell Cornification

The activation of p42/44 MAPK by PKC has been established in many cell types (30, 36). MAPK plays an integrating role in the signaling cascade. Activated MAPK can phosphorylate and then regulate the activity of numerous cellular signaling molecules, including cell-surface protein, cytoskeletal components, cytoplasmic kinase, and nuclear transcriptional factors (37). Thus, we further tested whether PMA-induced cell cornification occurs through an MAPK pathway, using the very specific MEK inhibitor PD98059. Confluent cultures of passage 1 human tracheobronchial epithelium were pretreated with PD98059 (50 µM) for 1 h and then treated with PMA. As shown in Figure 4, PMA treatment enhanced the phosphorylation of ERK-1 and ERK-2. However, pretreatment with PD98059 was able to prevent the enhancement. For the cell cornification study, cells were pretreated with various concentrations (50, 75, and 100 µM) of PD98059 for 1 h and then shifted to PMA plus the same concentration of PD98059 for 24 h. At concentrations of 50 and 75 µM, PD98059 had no effect on PMA-induced cell cornification (Figure 5) or change in cell morphology (Figure 6). PD98059 at the 100-µM level was toxic for the cells. These results suggest that PMA- induced cell cornification is not mediated by MAPK.

View larger version (14K): [in this window] [in a new window]   Figure 4.   Regulation of ERK activation by PMA and MEK inhibitor in human TBE cells. Passage 1 human TBE cells were maintained as described in MATERIALS AND METHODS. Before the PMA (5 ng/ ml) treatment, cultures were pretreated with MEK inhibitor, PD98059 (50 µM), or with DMSO alone as control. Cell lysates were prepared at indicated time intervals and 40 µg of total proteins were subjected to SDS-PAGE analysis and Western blot analysis with a monoclonal antibody specific to activated ERKs. Activated ERK1 and ERK2 migrated at the molecular weights of 44 and 42 kD, respectively. Uniform loading of proteins was assessed by probing the blot with an antibody against pan ERK1 and ERK2 (data not shown).

View larger version (43K): [in this window] [in a new window]   Figure 5.   Effects of MEK inhibitor on PMA-induced cross-linked envelope formation in human TBE cells. Passage 1 human TBE cells were maintained in a control culture condition as described in MATERIALS AND METHODS. At Day 6, cells were treated with PMA (5 ng/ml) or pretreated with MEK inhibitor PD98059 (50, 75, and 100 µM) for 1 h and then shifted to PMA plus the same concentration of PD98059. Cross-linked envelopes were quantified 24 h after the treatments. Data from triplicate dishes are expressed as means ± SD. PD98059 at 100 µM of concentration was toxic to the cells, so no data point is shown on the graph.

View larger version (84K): [in this window] [in a new window]   Figure 6.   Effects of MEK inhibitor on PMA-induced cell morphology changes. Experiments were performed as described in Figure 4. The pictures were taken 24 h after the treatments. (A) Control culture; (B) PMA treatment; (C) PMA plus 75 µM PD98059.

Effects of PKC and MAPK Inhibitors on PMA-Induced SPRR1 Gene Expression

Similar experiments were performed to elucidate the effects of these mediators on the regulation of PMA-enhanced SPRR1 gene expression. As shown in Figure 7, PMA induced a 2-fold increase in the level of SPRR1 mRNA. The PKC inhibitor BIM I completely abolished PMA-induced enhancement in SPRR1 mRNA. BIM I alone, however, had no stimulatory or inhibitory effect on SPRR1 mRNA level, a finding consistent with previous experiments with a different PKC inhibitor, sphingosin (11, 12). These findings suggest that the PKC-mediated pathway is involved in the regulation of PMA-dependent stimulation of SPRR1 gene expression.

View larger version (38K): [in this window] [in a new window]   View larger version (54K): [in this window] [in a new window]   Figure 7.   Effects of PKC and MEK inhibitors on PMA-induced SPRR1 gene expression. Passage 1 human TBE cells were maintained in a control culture condition as described in MATERIALS AND METHODS. At Day 6, cells were treated with PMA (5 ng/ ml, lane 2), BIM I (106 M, lane 4  ), and PD89059 (75 µM, lane 7  ), or pretreated with BIM (lane 3) and PD89059 (50 and 75 µm, lanes 5 and 6) for 1 h and then shifted to PMA plus the same inhibitors, respectively (lane 1, control RNA). Total RNA were harvested 24 h after the treatment. Northern blot hybridization (A) and quantification (B) were carried out as described in MATERIALS AND METHODS.

In contrast to the cell cornification event, PMA-dependent stimulation of SPRR1 mRNA was sensitive to the MEK1 and MEK2 inhibitor PD98059 (Figure 7). At a concentration of 50 µM, PD98059 partially attenuated the increase of SPRR1 mRNA induced by PMA. At 75 µM, PD98059 completely abrogated the increase by PMA. This inhibitor by itself also had no effect on the SPRR1 mRNA level (Figure 7). These results suggest that PMA enhances SPRR1 gene expression via PKC and then MAPK pathways.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

PMA is a potent inducer for both cell cornification and the expression of SPRR1 in conducting airway epithelial cells. Using passage 1 culture of human TBE cells, the effects of PMA on both events are PKC-dependent, as shown by the fact that a specific PKC inhibitor, BIM I, completely abrogated both the increase of cross-linked envelope formation and the enhanced level of SPRR1 mRNA by PMA. Further, another PKC activator, indolactom V, can also achieve a stimulation similar to that of PMA on cell cornification. These results are consistent with studies from other laboratories and our own previous work. Both Jetten (6) and Willey and colleagues (9), using PKC activators different from PMA, have observed a similar enhancement of cell cornification in airway epithelial cell cultures. Jetten further demonstrated that the event could be inhibited by a PKC inhibitor (6). In the case of SPRR1 gene expression, we have shown previously that sphingosin and the receptor downregulation approach (11) can attenuate PMA stimulation. Further, we have demonstrated at the genomic footprinting level that both TPA-responsive element (TRE) and TRE-like motifs at the 5'-flanking region were responsible for DNA-protein interaction during the stimulation of transcription by PMA (12). These findings strongly support PKC-dependent signaling as a major pathway in the regulation of cell cornification and SPRR1 gene expression by PMA.

To further elucidate the downstream signaling pathway after PKC activation by PMA, we found a divergence in the regulation of cell cornification and SPRR1 gene expression. For the latter, the stimulation could be blocked by a very specific MEK1/MEK2 inhibitor, PD98059 (39), whereas the cornification was not affected. This is the first demonstration that expression of squamous-cell differentiation is regulated by a diverse mechanism. The activation of p42/ p44 MAPK by phorbol ester has been observed in many cell types. A pathway from PKC-Ras-Raf-MEK1/MEK2 to ERK1/ERK2 is also well established (30). However, our finding that PD98059 did not block the cornification induced by PMA indicates that this induction does not take place through the MAPK pathway. It is still unclear which pathway is followed after PKC activation in the regulation of cell cornification. In a separate study, we demonstrated that neither new protein nor new RNA synthesis is needed for the cell cornification induced by PMA (data not shown). This suggests that it is more likely that PMA induces cell cornification by acting on existing cellular substrates, such as the interaction between the cross-linking enzyme transglutaminase I and cornified envelope substrates such as involucrin, loricirn, SPRR1, etc., rather than through the initiation of new gene expression for the event.

For SPRR1 gene expression, the downstream signaling pathway in the regulation is clearer than the cell cornification event, inasmuch as the expression is sensitive to MAPK inhibitor. Our previous experiments on the promoter of human SPRR1 gene have shown that PMA-induced SPRR1 gene expression is c-Jun dependent. In transient transfection studies, it has been found that overexpression of c-Jun and PMA treatment synergistically stimulate the SPRR1 promoter activity more than 40-fold. Further, we observed that c-Jun expression was increased shortly after PMA treatment in human TBE cells (12). Because the activation of MAPK by PKC (36, 37), upregulation of c-Jun expression, and activation of c-Jun by MAPK have been shown in many cell types (37), it is more likely that PMA-stimulated SPRR1 gene expression occurs through the MAPK pathway. This theory is confirmed by the current study, which shows that a specific MEK1/MEK2 inhibitor, PD98059, completely blocked PMA-induced upregulation of SPRR1 mRNA. The signaling pathway for phorbol ester-induced activation of MAPK has been studied in detail in COS1 cells and NIH3T3 cells. There are at least two pathways, depending on the subtypes of PKC activated, involved in the activation of p42/44 MAPK. PKC- activates p42/44 MAPK through the Ras-Raf-MEK-ERK pathway. However, PKC activates ERK through a Ras-independent Raf pathway (30). Because there is no information as to which PKC isoenzyme was activated, further studies are needed to elucidate which signaling pathway is involved in PMA-induced activation of MAPK in airway epithelium. However, based on this and previous studies, we conclude that PKC, MAPK, and c-Jun are involved in PMA-enhanced SPRR1 gene expression.

Although cell cornification in vivo is rare, it can happen in airway epithelium as a scar in the region under an extreme condition (1). One of the critical steps in cell cornification is the activation of transglutaminase type 1 to initiate cross-linked formation for various substrates, such as keratins, involucrin, loricrin, and small proline-rich proteins. The present study has demonstrated that regulation of the synthesis of these substrates and the enzymatic activation may involve diverse signaling pathways. This lesion should be studied further in vivo to outline the critical signaling step in the regulation of airway squamous epithelial cell differentiation.

In summary, we have demonstrated that both cell cornification and SPRR1 gene expression by PMA are regulated through a common pathway, but one that is divergent. The data suggest that PKC-activation is the common pathway shared by these two processes. However, the PMA-induced upregulation of SPRR1 gene expression is through an MAPK dependent pathway, whereas PMA-induced cell cornification takes place through an MAPK-independent pathway. Which isoenzyme of PKC is involved and how MAPK is activated by PMA remain subjects for future experiments.