Exposure of distal bronchiolar region to various toxicants and pollutants suppresses Clara cell differentiation marker expression and greatly enhances the induction of squamous cell differentiation (SCD). Here, we demonstrate for the first time phorbol 13-myristate 12-acetate (PMA)-inducible expression of SCD markers,SPRRs, in Clara-like H441 cells. The transcriptional stimulation of human SPRR1B expression is mainly mediated by a −150- to −84-bp region that harbors two critical activator protein (AP)-1 sites. In unstimulated cells, the −150- to −84-bp region is weakly bound by AP-1 proteins, mainly JunD and Fra1. However, PMA prominently induced the binding of JunB and Fra1. Consistent with this, overexpression of wild-type Jun proteins upregulated theSPRR1B promoter activity. Conversely, a c-jun mutant suppressed both basal and PMA-inducible reporter gene expression. Intriguingly, overexpression of fra2 suppressed PMA-inducible reporter activity, whereas fra1 significantly enhanced basal level activity, indicating an opposing role for these proteins in SPRR1B expression in a manner similar to that observed in proximal tracheobronchial epithelial cells (BEAS-2B clone S6). Interestingly, unlike in S6 cells, a catalytically inactive c-Jun NH2-terminal kinase (JNK) 1 mutant significantly reduced the PMA-inducible SPRR1B promoter activity in H441 cells. Thus either temporal expression and/or spatial activation of AP-1 proteins by JNK1 might contribute to the induction of SCD in Clara cells.
- small proline rich proteins
- airway epithelium
- transcriptional regulation
- mitogen-activated protein kinases
- c-Jun NH2-terminal kinase 1
- activator protein 1
PHORBOL 13-MYRISTATE 12-ACETATE (PMA), a stable functional analog of diacylglycerol, is produced in vivo by hydrolysis of phospholipids, which induces diverse biological responses. PMA mainly activates several protein kinase C (PKC) isoenzymes, which in turn initiate a signaling cascade to regulate the expression of genes that are involved in cell growth and differentiation in response to environmental stimuli including cellular injury repair, inflammation, and acute phase response. Clara cells are the progenitor cell type for the distal bronchiolar epithelium during injury and repair (16). Administration of PMA, which induces acute and progressive lung injury (12), downregulates the expression of human surfactant protein gene expression in both human fetal lung implants and in Clara-like bronchiolar epithelial cell line H441 (9, 13, 15, 19,20). PMA also potently induces squamous cell differentiation (SCD) in tracheobronchial epithelial (TBE) cells in vivo and in vitro. Previous studies have shown a close relationship between an early induction of human small proline-rich protein type I (SPRR1) and SCD in TBE cells (27, review). Tobacco smoke, which potently induces SCD in airway epithelium, enhances the expression ofSPRR1 in nasal (28) and bronchial epithelium (22). Subsequently, it was shown that PMA (1,21) and tobacco smoke (22) induce expression ofSPRR1 (now referred to as SPRR1B) in TBE cells, which occurs primarily at the transcriptional level and is mediated by the TRE sites and activator proteins (AP)-1.
The distal bronchiolar region represents a major target for pulmonary toxicity induced by various environmental toxicants and pollutants (16). Several studies have demonstrated that the exposure of animals to toxicants, such as naphthalene, selectively induces cellular injury in distal bronchiolar region (25, 29). For example, naphthalene at lower levels (50–100 mg/kg body wt) induces Clara cellular injury in the distal airway region, whereas proximal airways require a substantially higher dose (>300 mg/kg) (17). Later studies attributed this difference to a higher level of cytochrome P-450 isoenzyme 2F2, which converts naphthalene to an active metabolite, 1R, 2S-naphthalene, in distal airway region compared with proximal airways (3). Conversely, the content of glutathione, which plays a protective role in cellular detoxification, is higher in upper airways compared with distal bronchiolar region (18). Together these studies indicate certain differences in the mechanisms governing cellular injury and repair process in upper and distal airway regions. Naphthalene suppresses the expression of Clara cell differentiation (CCD) markers and greatly enhances the expression of squamous cell functions (29). Moreover, exposure of mice to naphthalene and tobacco smoke greatly enhances the expression SPRR1B in distal bronchiolar region (Reen Wu, University of California at Davis, Davis, CA; personal communication). However, the exact molecular mechanisms governing the regulation of cell proliferation and differentiation in response to injury, in particular the induction of SCD, in the distal bronchiolar region, are not clearly understood. Based on certain differences involved in injury and repair mechanisms, we hypothesized that the induction of SCD in the distal bronchiolar region is regulated by a mechanism different from those that operate in the proximal airway epithelium. In the present study, we used Clara-like bronchiolar epithelial cell culture (H441) as a model system to study the regulation of CCE precursor protein gene expression and to understand the molecular mechanisms involved in the induction of SCD in the distal bronchiolar region. Here, we present for the first time evidence for a PMA-inducible expression ofSPRRs in distal bronchiolar cells H441. Promoter analysis indicates that, similar to proximal TBE cells (S-6), PMA-stimulated SPRR1B expression in H441 cells requires the same TREs (PMA-responsive elements or AP-1 sites) and combination of AP-1 proteins. However, unlike in S6 cells, c-Jun NH2-terminal kinase (JNK) 1, a mitogen-activated protein kinase (MAPK), is critical for PMA-inducible SPRR1Bexpression in H441 cells. Thus the activation of different signaling pathways might contribute to the induction of SCD in the distal bronchiolar region.
MATERIALS AND METHODS
Cell culture, Northern blot, RT-PCR, and Western blot analyses.
NCI-H441 (Clara-like bronchiolar pulmonary adenocarcinoma) cell line was obtained from ATCC. All cells were maintained in RPMI culture medium supplemented with 5% serum, 1% streptomycin and penicillin, gentamicin (250 ng/ml), and fungizone (125 ng/ml). Cells were treated with either vehicle (DMSO) or PMA (25 ng/ml) for the indicated time periods. Total RNA (20 μg/lane) was separated on a 1.2% agarose gel and transblotted. Northern blot analysis was carried out using32P-labeled cDNAs of SPRR1B as previously described (31). RT-PCR was performed as previously described (14). Briefly, total RNA (750 ng) was reverse transcribed into cDNA, and PCR amplification was performed with an aliquot of cDNA using gene-specific primer pairs (see Table1). The indicated number of cycles were employed to amplify the product in a linear range. The amplified cDNA fragments were separated and quantified with the Gel Doc 2000 System (Bio-Rad). Western blot analysis of cellular extracts isolated from untreated and PMA-treated H441 cells was performed using polyclonal antibodies of human SPRR1B (10).
Transient transfections and reporter gene assays.
The wild-type (wt), dominant negative (dn), and constitutively active (ca) expression vectors were kindly provided by the following: the dn-mitogen-activating protein kinase kinase (MAPKK) 1 and ca-MAPKK1 cloned in pCMV vector, Dennis Templeton, Case Western Reserve University, Cleveland, OH; dn-JNK1 cloned in pCDNA3 vector, Roger Davis, Howard Hughes Medical Institute, Rochester, NY; dn-extracellular signal-regulated kinase (ERK) 1 and dn-ERK2 cloned in pCEP4 vector, Melanie Cobb, University of Texas Southwestern Medical Center, Dallas, TX; wt-c-Jun and dn-c-Jun (has mutations in transactivation domain) cloned in pCMV vector, Michael Birrer, National Cancer Institute, Bethesda, MD; wt-JunB and wt-JunD cloned in SR-alpha vector, Michael Karin, University of California at San Diego, San Diego, CA; wt-Fra1 cloned in pCMV vector, Eugene Tulchinsky, Institute of Cancer Biology, Copenhagen, Denmark; and wt-Fra2 cloned pCMV vector, Donna Cohen, Australian National University, Canberra, Australia; dn-Raf (Raf-C4) cloned in pRSV vector (Stephen Ludwig, Institut für Medizinische Strahlenkunde und Zellforschung, Würzburg, Germany) were obtained. Dn-Ras (Ras-N17) and ca-Ras (Ha-Ras-V12) were generated as previously described (31). Several deletion and site-directed mutational fragments of the −150 to +12-bpSPRR1B promoter fused to the luciferase (Luc) gene as described previously (21). DNA transfections were performed using a Fugene transfection reagent (Roche Biochemical). Cells were grown on 48-well plates at 70–80% confluence and then transfected with 100 ng of promoter construct, 50 ng of CMV-β-galactosidase (β-gal) DNA, and 50–200 ng of empty or expression plasmid vectors. After 18–20 h posttransfection, cells were treated with either DMSO or PMA (25 ng/ml) for 14–16 h. Cells were lysed, and Luc activity was measured using a commercially available kit (Promega). Luc activity of individual samples was normalized against β-gal activity and/or total protein as described previously (31). Luc activity for every construct was analyzed in duplicate samples, and all experiments were repeated three to four times.
Electrophoretic mobility shift assay.
Nuclear extracts from DMSO- or PMA-treated H441 cells were prepared, and electrophoretic mobility shift assay (EMSA) was performed as described previously (14) with 0.1–0.5 ng of32P-end-labeled consensus AP-1 site or −150- to −84-bpSPRR1B promoter fragment (for sequence see Table2). For supershift analysis, nuclear extracts were mixed with 1–2 μg of c-Jun (sc-45X), JunB (sc-46X), JunD (sc-74X), c-Fos (sc-7202X), Fra1 (sc-605x), and Fra2 (sc-604X) antibodies (Santa Cruz Biotechnology) and incubated on ice for 1–2 h before the labeled DNA probe was added.
In vitro kinase assays and immunoblot analysis.
H441 cells were cultured to 70–80% confluence and treated with PMA (25 ng/ml) for indicated time periods, and assays were performed as previously described (31). Briefly, 200 μg of cellular protein were immunoprecipitated using 0.4 μg antibodies derived against JNK1 (C-17) or p38 (C-20) kinases (Santa Cruz Biotechnologies) that were conjugated to protein A-Sepharose (Pharmacia). The immunoprecipitates were washed extensively, and the kinase activities of JNK1 and p38 were determined as previously described (31). Active ERK1/2, p38, and JNK1 immunoblot analysis was carried out using the phospho-specific antibodies as previously described (31). Total ERK, p38, and JNK1 content of the samples was determined by immunoblot analysis using ERK, p38, and JNK1 antibodies, respectively. Protein loading of the membrane was assessed by Western analysis using γ-tubulin antibody (Sigma, T 6557).
Data are expressed as the means ± SE. The StatView program was used to perform analysis of variance between different samples. Statistical significance was accepted at P < 0.05. All assay samples were performed in duplicate, and each experiment was repeated at least two times.
PMA upregulates SPRR expression in H441 cells.
Previous studies have demonstrated a close association between the expression of SPRR1 and induction of airway SCD (27). To understand the molecular mechanisms of SCD in the distal bronchiolar region, we treated H441 cells with either DMSO (vehicle) or PMA (25 ng/ml) for different time periods as indicated. Total RNA was isolated, and Northern blot analysis was performed with comparable amount of RNA from each sample. As shown in Fig.1 A, SPRR1B mRNA levels are undetectable in the DMSO-treated control group (lane 1). However, by 3 h, PMA strongly enhanced theSPRR1B mRNA (lane 2), which reached a maximum by the 6-h time period and returned to lower levels by 14 h. All lanes had comparable amounts of 28S RNA, a housekeeping gene (Fig.1 A, bottom). A similar pattern of SPRR1B protein expression was observed after PMA treatment. As shown in Fig.1 B, H441 cells displayed a very low basal level of SPRR1B (lane 1), but PMA significantly enhanced the protein levels (lane 2).
To distinguish SPRR1B expression from its orthologSPRR1A, which shows significant sequence homology with the coding region of 1B, we analyzed the SPRR1Bmessage levels employing a semiquantitative RT-PCR with gene-specific primers (see Table 1). A good corroboration was observed between the results of Northern blot and RT-PCR. PMA induced the expression ofSPRR1B message levels in H441 (Fig. 1 C) similar to normal transformed bronchial epithelial cells HBE1 and S6 (1,21). We next analyzed the expression of other SPRRfamily members (SPRR1A, SPRR2A, and SPRR3) in H441 cells. PMA significantly induced SPRR1A andSPRR2A but not SPRR3 message levels in H441 cells. Together, these results indicate that PMA differentially induces the expression of SPRRs in Clara-like bronchiolar epithelial cells.
Induction of SPRR1B expression in H441 cells is regulated mainly at the transcription level.
Previous studies have shown that PMA downregulates the CCD marker expression, surfactant protein (SP)-A and SP-B, in H441 cells at the transcriptional and posttranscriptional levels, respectively (9,13, 19, 20). In the present study, we analyzed the regulation of SPRR1B in H441 cells to understand the molecular mechanisms (transcriptional and/or posttranscriptional) governing the induction of SCD in distal bronchiolar region. We mainly focused on SPRR1B regulation, as our previous studies and those of others have demonstrated a close association ofSPRR1B expression with airway SCD both in vivo and in vitro (1, 21, 26). H441 cells were pretreated with actinomycin D (10 μg/ml) for 30 min before PMA exposure. RNA was isolated 6 h later, and SPRR1B message levels were analyzed by RT-PCR. As shown in Fig. 2 A, PMA significantly induced the SPRR1B message levels (lane 2). However, pretreatment of cells with actinomycin D before PMA exposure significantly reduced inducible SPRR1B message levels (lane 4). To analyze whether PMA has any effect onSPRR1B mRNA stability, we treated cells with PMA for 6 h and then with actinomycin D. Cells were harvested at indicated time points thereafter. As shown in Fig. 2 B, actinomycin D did not significantly affect the message levels stimulated by PMA. These results indicate that PMA-inducible expression of SPRR1B in H441 cells is regulated at the transcriptional level.
TREs located between the −150- and −84-bp promoter modulate PMA-inducible SPRR1B expression in H441 cells.
To characterize the regulatory elements involved in PMA-inducibleSPRR1B expression, several promoter mutants ofSPRR1B linked to the Luc gene (Fig.3 A) were transiently transfected into H441 cells. We have mainly focused on the region from −150 to −84 bp, as it contains the motifs necessary for basal and PMA-inducible SPRR1B regulation in TBE cells (21). Luc expression in the presence (dark bars) or absence of PMA (light bars) was analyzed. As shown in Fig.3 B, the 113-Luc construct, which bore the proximal TRE (−109), displayed basal level activity, whereas PMA significantly upregulated it. The 150-Luc construct, which contains both the proximal and distal TREs, exhibited both basal enhanced and a high-level PMA-inducible activity. As shown in Fig. 3 C, mutation of both TREs [double mutant (DM)] ablated both basal and PMA-inducible expression. Interestingly, mutation of either TRE alone did not have a significant effect on both basal and PMA-stimulated gene expression (data not shown). Interestingly, mutation of the promoter region between the two TREs (IN) also significantly reduced the basal level activity indicating the importance of these flanking sequences in gene regulation. These results are consistent with our previous studies that demonstrated a critical role for both these TREs in regulating PMA-inducible expression of SPRR1B (21).
Analysis of AP-1 binding to the consensus AP-1 site and the SPRR1B promoter.
The above data indicated that the TREs of SPRR1B promoter are necessary for PMA-stimulated expression in H441 cells. We therefore examined the patterns of AP-1 proteins binding to the consensus AP-1 site (Fig. 4). Nuclear extracts were isolated from untreated and PMA-treated H441 cells incubated with a32P-labeled consensus AP-1 site (Fig. 4 A). A basal-level AP-1 protein binding was observed in the unstimulated cells (lane 1), which was significantly enhanced with PMA treatment (lane 2). We next examined the composition of AP-1 protein complex in untreated (Fig. 4 B, lanes 2–7) and PMA-treated (Fig. 4 B, lanes 9–14) H441 cells using antibodies specific for the individual Jun (c-Jun, JunB, and JunD) and Fos (c-Fos, Fra1, and Fra2) proteins. PMA significantly enhanced the binding of c-Jun, JunB, JunD, Fra1, and Fra2, but not c-Fos, as revealed by a supershift of their complexes with specific antibodies. Thus PMA enhanced the binding of Jun and Fos family members to a consensus AP-1 site similar to the patterns observed in HBE1, S6, and A549 cells (14).
We next characterized the composition of the protein complex binding to the −150- to −84-bp region of the SPRR1B promoter, henceforth SPRR-TRE, that contains two critical TREs. As shown in Fig.5 A, vehicle-treated H441 nuclear extracts (lanes 1 and 7) formed a DNA-protein complex that was enhanced significantly after PMA treatment (lane 8). The specificity of the DNA-protein complex was demonstrated by a competitive inhibition of complex formation by the unlabeled SPRR-TRE (self, lane 3) and consensus AP-1 site (AP-1, lane 4). However, unlabeled DM did not block the complex formation (lanes 5 and 6). Furthermore, mutation of TRE sites (for sequence, see Table 2) completely blocked AP-1 complex formation (data not shown). We next characterized the composition of the AP-1 protein complex binding to the SPRR1Bpromoter using specific antibodies as in Fig. 5 B. Before the PMA treatment, cells displayed binding of JunD and Fra1 (lanes 4 and 5). However, treatment of cells with PMA predominantly induced binding of JunB and Fra1 (lanes 8–14). A slight enhancement of Fra2 binding was also noticed after PMA treatment (lane 13). Preincubation of nuclear extracts with normal rabbit serum showed no supershifting of the bands (data not shown). These results indicate that the AP-1 dimers, consisting of JunD and Fra1, bind to SPRR-TRE in the unstimulated state, whereas JunB and Fra1 bind to it upon PMA treatment.
AP-1 proteins differentially regulate SPRR1B promoter activity.
Band shift profiles indicated a distinct pattern of AP-1 binding to SPRR-TRE compared with the consensus AP-1 site. Therefore, we used a panel of wt and/or dn mutants of the Jun (c-jun,junB, and junD) and Fos(fra1 and fra2) expression plasmids to determine the roles for these proteins in the regulation ofSPRR1B. We did not study the role of c-Fos, because band shifts studies did not reveal any significant binding of c-Fos to either consensus AP-1 site or SPRR-TRE in both untreated and PMA-treated cells. Cells were transiently transfected with 150-Luc reporter plasmid along with the indicated expression vectors at comparable molar quantities, and Luc activity was analyzed. As shown in Table 3, cotransfection of wt c-jun, junB, and junD expression vectors robustly enhanced the promoter activity to levels comparable with those observed with PMA treatment. Conversely, overexpression of c-Jun mutant with a defective in transactivation domain strongly inhibited both basal and PMA-stimulated SPRR1B expression. As shown in Fig. 5 B, Fra1 binds to the SPRR1Bpromoter predominantly, whereas a slight increase in Fra2 binding was noticed after PMA exposure. Therefore, we analyzed the role for Fra1 and Fra2 in SPRR1B promoter regulation. Overexpression offra2 strongly suppressed PMA-stimulated Luc expression driven by SPRR1B promoter. Conversely, overexpression offra1 upregulated the basal level activity, whereas it had no significant effect on PMA-stimulated promoter activity (Table 3). Together, these results demonstrate that AP-1 dimers, with distinct composition, upregulate SCD in distal bronchiolar region. More recently, we showed that binding of Fra2 to the SPRR-TRE correlates with a loss of SPRR1B expression in A549 cells (14). In contrast, overexpression of Fra1 strongly upregulates SPRR1B promoter-driven reporter gene expression in A549 cells (14). These results indicate that Fra1 and Fra2 exert opposing effects on SPRR1B promoter in H441 cells. They also indicate that PMA-inducible expression of SPRR1Bin distal bronchiolar cells is regulated by TRE sites and AP-1 proteins in a manner similar to that observed in proximal TBE cells, S6 and HBE1 (14).
Ras regulates SPRR1B gene expression through Raf-1 and MAPKK kinase 1.
To understand the signaling pathways that control PMA-inducible expression, we examined the role of Ras-Raf MAPK pathway. As said earlier, PMA activates PKC isoforms that in turn activate Ras, one of the downstream targets of PKC (30, 33). To determine whether PMA upregulates SPRR1B expression through Ras, we cotransfected cells with ca-Ras or mutant dn-Ras expression vectors along with reporter construct. After 24 h, cells were treated with either PMA or vehicle, and the Luc expression was monitored. Expression of dn-Ras suppressed PMA-stimulated activity very significantly (Fig. 6 A), whereas it had no effect on basal expression. Conversely, overexpression of ca-Ras significantly stimulated promoter activity (∼2.5-fold) compared with vector-transfected controls (comparebar 1 with 5). We next investigated whether Raf-1, the downstream target of Ras (30), is required for gene induction (Fig. 6 A). Indeed, dn-c-Raf significantly suppressed PMA-stimulated, but not the basal-level, SPRR1Bexpression (compare bars 2 and 7). Together, these results indicate that PMA-activated Ras and Raf are critical for activating SPRR1B promoter. Ras also activates MAPKK kinase (MAPKKK) 1. Expression of dn-MAPKKK1 did not have any effect on basal gene expression, whereas it had a very significant effect (nearly 50% reduction) on PMA-stimulated promoter activity (Fig. 6 B). On the other hand, expression of ca-MAPKKK1 alone strongly augmented (∼4-fold) the Luc expression (Fig. 6 B).
MAPKK1, but not ERK1/2 MAPKs, regulates SPRR1B promoter in H441 cells.
Recently, we showed that PMA-stimulated SPRR1B expression in BEAS-2B cells is mainly regulated by a MAPKK1/ERK-like MAPK pathway. However, ERK1/2, JNK, and p38 MAPKs did not appear to participate in this pathway (31). We therefore focused our studies on the latter three (ERK, JNK, and p38) MAPKs and used both pharmacological and/or gene-specific dn mutants to evaluate whether PMA-inducible expression of SPRR1B in Clara-like H441 cells is mediated by the same signaling pathways as in proximal TBE cells. PD-98059, which prevents the activation of MAPKK1/2 (7), strongly suppressed both the basal (∼50%) and PMA-stimulated (∼100%) reporter gene expression (Fig.7 A). These results have been further confirmed with UO-126 (data not shown), which specifically inhibits the function of activated MAPKK1 (7). Consistent with this, expression of a dn-MAPKK1 robustly suppressed PMA-stimulated reporter gene expression; conversely, ca-MAPKK1 significantly enhanced it (Fig. 7 B). Furthermore, PD-98059 slightly reduced basal and very significantly suppressed PMA-stimulated endogenousSPRR1B expression (data not shown). Thus MAPKK1 regulates both basal and PMA-stimulated expression of SPRR1B. We next examined the role of ERK1 and ERK2, the well-known downstream targets of MAPKK1, in SPRR1B expression. As shown in Fig.7 C, overexpression of a dn-ERK1 or dn-ERK2 either alone or in combination (data not shown) did not suppress PMA-stimulated reporter gene expression. Intriguingly, treatment of H441 cells with PMA enhances the phosphorylation of ERK1/2 proteins (three- to fivefold). The ERK1/2 phosphorylation rapidly increased within 5 min and persisted at later periods (Fig. 7 D). Together, these results indicate that the MAPKK1 pathway regulates PMA-stimulatedSPRR1B expression. Paradoxically, ERK1 and/or ERK2 do not participate in it.
JNK1, but not p38 MAPK, regulates SPRR1B promoter in H441 cells.
The JNK and p38 MAPK family members are activated in response to cellular injury, ultraviolet irradiation, inflammatory cytokines, and various environmental stresses (8). Therefore, we next examined a role for p38 and JNK MAPKs in SPRR1B promoter regulation. To examine the role of p38 pathway, we used a chemical inhibitor, SB-202190 (SB). H441 cells were pretreated with SB (5–20 μM), and endogenous SPRR1B mRNA levels were measured by RT-PCR analysis (Fig.8 A). SB did not significantly suppress the PMA-stimulated mRNA expression of SPRR1B. Consistent with this, SB did not significantly inhibit either the basal or PMA-stimulated SPRR1B promoter-driven reporter gene expression (Fig. 8 B). Intriguingly, the kinase activity of p38, as measured by the phosphorylation of its substrate, activating transcription factor (ATF)-2, was significantly increased after PMA treatment (Fig. 6 C). Kinase activity increased onefold within 5 min of treatment and reached peak values (sixfold) after 15 min of treatment. The kinase activity remained elevated, below the peak level, even after 3 h. Together, these results indicate that the p38 MAPK pathway, though functionally active, does not appear to regulate PMA-stimulated SPRR1B expression in the Clara cell type.
We next examined the role of JNK1 in the regulation ofSPRR1B promoter (Fig. 9). Expression of dn-JNK1 mutant significantly diminished PMA-stimulated expression (Fig. 9 A). JNK1 and JNK2 activation was also confirmed by immunoprecipitation with isoform-specific antibodies followed by Western blot analysis with phospho-JNK-specific antibodies. As shown in Fig. 9 B, PMA treatment significantly enhanced phosphorylation of JNK1 and JNK2 proteins in H441 cells. Consistent with this, enzyme activity of JNK1/2 was also enhanced after PMA treatment. PMA significantly increased JNK1 kinase activity (fourfold) that persisted over a longer time period (Fig. 9 C). Together, these results highlight the importance of JNK1 in regulation of PMA-stimulated SPRR1B expression in H441 cells (Fig. 10).
In response to injury caused by various carcinogens, toxicants, and pollutants, airway epithelial cells (proximal and distal) lose their normal secretory functions and express squamous and keratinizing markers (16). Induction of SCD in nonsquamous airway epithelium is initially thought to be a protective response, acting as a barrier against toxicants and pollutants; however, if not properly restored, this phenomenon might lead to airway epithelial cell transformation and bronchial carcinogenesis (11). An understanding of the molecular events controlling the SCD process will help us in delineating the pathways involved in cellular injury and repair processes. It also provides an insight into the toxicant-induced airway disease and bronchial carcinogenesis. Clara cells are nonciliated epithelial cells that play an important role in respiratory function by expressing surfactants and a 10-kDa Clara cell protein, CC10 (16). The present study demonstrates that PMA selectively induces the expression of SCD markers such asSPRRs (1A, 1B, and 2A, but not 3) in Clara-like bronchiolar epithelial cells. These results are in contrast with the previous studies that demonstrated the downregulation of CCD markers, such as CC10 and surfactant expression in H441 cells, upon exposure to PMA (9, 13, 19, 20). Similarly, naphthalene and furan 4-ipomeanol induce cellular injury and suppress the expression of CCD markers, such as CC10 and cytochrome P-450, and also greatly enhance the expression of squamous cells (∼70–90%) in the distal bronchiolar region (23). The exact role for SPRRs in the induction of SCD in Clara cells is unclear. However, it is well established that SPRRs act as cross-bridging proteins that participate in a cross-linking process of several CCE precursor proteins, such as loricrin (constituting 50–90% of CCE proteins) and involucrin (4, 24). It is worth noting that a recent study demonstrated the actual participation of SPRR1B in the cornification of proximal TBE cells (5). Therefore, we speculate that expression of SPRRs is an essential feature of SCD of distal bronchiolar cells and plays a very important role in providing a physical and chemical barrier against the environment. In support of this notion, a selective inducible expression ofSPRRs, but not involucrin, was noticed in malignant keratinocytes that were induced to differentiate (32), underscoring an important role for SPRRs in epithelial cell differentiation. Moreover, Chinese hamster ovary cells, a nondifferentiating cell type, express SPRR1 at specific stages of the cell cycle indicating that SPRRs may play a novel role in cell cycle progression and growth arrest (26). Thus, transcriptional regulation of SPRR expression may provide an additional insight into molecular mechanisms governing the cellular differentiation of distal bronchiolar cells in response to toxic stimuli.
Abnormal expression and/or activation of AP-1 (Jun/Fos) proteins in response to various growth factors and toxicants had been implicated in the development of various diseases and epithelial cell transformation (2). The Jun family (c-Jun, JunB, and JunD) proteins can either homo- or heterodimerize with the Fos family (c-Fos, Fos-B, Fra1, and Fra2), bZIP transcription factors (e.g., ATF/cAMP response element binding protein, Maf/Nrf family members), as well as bHLH ZIP proteins (e.g., myoD and upstream stimulatory factor), resulting in differential DNA binding and transactivation (2). Subsequent AP-1 binding to the TREs regulates the transcription of a variety of genes that are involved in cell growth and differentiation and the injury-repair process, depending on cellular and promoter context (2). Transcriptional analysis indicates that two functional TREs and AP-1 proteins mediate PMA-inducible SPRR1B expression in H441 cells. Moreover, the composition of AP-1 protein complex (Jun/Fra1 dimers) plays an important role in regulation of SPRR1Bexpression in H441 cells, in a manner similar to that observed in proximal S6 cells. PMA stimulates both Fra1 and Fra2 binding to the consensus AP-1 or TRE site (Fig. 4 B), whereas Fra1 more prominently binds to the SPRR1B promoter compared with Fra-2 (Fig. 5 B). These results emphasize the importance of cellular and promoter context, flanking residues such as GT/CAC and ETS-like motifs, on the regulation of AP-1-dependent gene expression. Indeed, mutation of these sites significantly reduced basal-level promoter activity (Fig. 3 C). Thus it is conceivable that both the activation and proximal availability of Fra1 and Fra2 with other proteins such as Jun members and/or cell type-specific factors might be involved in the regulation ofSPRR1B expression in airway epithelial cells.
It is well established that PMA activates PKC isoenzymes, which in turn initiate a signaling cascade to stimulate expression of genes involved in cell growth and differentiation in different cell types. This is mediated by three well-studied distinct MAPKs, ERK, JNK, and p38 (6). ERK1 and ERK2 are mainly activated in response to growth stimuli, whereas JNK/SAPK and p38 are activated by cellular stress and cytokine inflammation and can participate in differentiation and cell death (6). In turn, these kinases activate transcription factors such as Elk-1, CCAAT/enhancer binding protein (C/EBP), C/EBP-homologous protein, c-Jun, and ATF-2 to modulate both cell type- and stimulus-specific target gene transcription (6). Recently, we demonstrated that a MAPKK1-ERK-like kinase pathway regulates PMA-stimulatedSPRR1B expression in TBE cells, whereas ERK1 and ERK2, JNK1/SAPK, and p38 had no effect (31). Consistent with this, the present study demonstrates an involvement of MAPKK1 in PMA-stimulated SPRR1B transcription in H441 cells; however, ERK1/2 do not participate in such a process. Interestingly, dn-JNK1 mutant robustly suppressed PMA-inducible SPRR1B expression in H441 cells (Fig. 9), indicating a regulatory role for JNK1 in the induction of SCD in distal bronchiolar cells. The fact that both MAPKK1 inhibitors and JNK1 mutant suppressed the PMA-stimulatedSPRR1B promoter activation suggests the existence of either other ERK-related kinase(s) or cross-talk between MAPKK1 and JNK1 pathways that might regulate gene expression. Our data (Fig. 6) clearly support a role for Ras in SPRR1B regulation. Suppression of PMA-stimulated SPRR1B promoter activity by c-Raf and MAPKKK1 mutants indicates that Ras mediates its effects in part through the activation of c-Raf and MAPKKK1. However, in S6 cells, PMA-stimulatedSPRR1B promoter regulation by Ras does not require c-Raf but is mediated by MAPKKK1 (31). Thus activation ofSPRR1B expression, although regulated by a similar set of AP-1 proteins, may be differentially activated by a unique set(s) of MAPKs in proximal and distal airway epithelial cells.
The authors thank all the scientists who provided us with various expression vectors used in this study. We thank Reen Wu and Steven Kleeberger for helpful comments on this study.
This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-58122 and a pilot project from National Institute of Environmental Health Sciences Center Grant ES-03819 to S. P. M. Reddy. We acknowledge the Johns Hopkins Urban Environmental Center for use of its core facilities.
Address for reprint requests and other correspondence: S. P. M. Reddy, Johns Hopkins Univ., Dept. of Environmental Health Sciences, Div. of Physiology, Rm. W7006, 615 No. Wolfe St., Baltimore, MD 21205 (E-mail:).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2002 the American Physiological Society