©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Evidence for the Involvement of Retinoic Acid Receptor RAR-dependent Signaling Pathway in the Induction of Tissue Transglutaminase and Apoptosis by Retinoids (*)

(Received for publication, July 13, 1994; and in revised form, December 20, 1994)

Li-Xin Zhang (1) Kevin J. Mills (1) Marcia I. Dawson (2) Steven J. Collins (3)(§) Anton M. Jetten (1)(¶)

From the  (1)Cell Biology Section, Laboratory of Pulmonary Pathobiology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, the (2)Bio-Organic Chemistry Laboratory, SRI International, Menlo Park, California 94025, and the (3)Program in Molecular Medicine, Fred Hutchinson Cancer Center, Seattle, Washington 98104

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In this study, we show that all-trans-retinoic acid (RA) is a potent inducer of tissue transglutaminase (TGase II) and apoptosis in the rat tracheobronchial epithelial cell line SPOC-1. We demonstrate that these cells express the retinoid receptors RARalpha, RAR, and RXRbeta. To identify which of these receptors are involved in regulating these processes, we analyzed the effects of several receptor-selective agonists, an antagonist, and a dominant-negative RARalpha. We show that the RAR-selective retinoid SRI-6751-84 strongly increased TGase II expression at both the protein and mRNA levels, whereas the RXR-selective retinoid SR11217 had little effect. The RARalpha-selective retinoid Ro40-6055 was also able to induce TGase II, whereas the RAR-selective retinoid CD437 was inactive. The induction of TGase II by the RAR-selective retinoid was completely inhibited by the RARalpha-antagonist Ro41-5253. Overexpression of a truncated RARalpha gene with dominant-negative activity also inhibited the induction of TGase II expression. The increase in TGase II is associated with an induction of apoptosis as revealed by DNA fragmentation and the generation of apoptotic cells. We demonstrate that apoptosis is affected by retinoids in a manner similar to TGase II. Our results suggest that the induction of TGase II expression and apoptosis in SPOC-1 cells are mediated through an RARalpha-dependent signaling pathway.


INTRODUCTION

Transglutaminases (EC 2.3.2.13, TGases) (^1)are Ca-dependent enzymes catalyzing the formation of (-glutamyl)lysine cross-links between polypeptide chains(1) . Several members of this gene family have been identified including the type I (epidermal) TGase (2, 3) and type II (tissue) TGase(4, 5) . TGase I is induced during squamous differentiation and plays a role in the formation of the cross-linked envelope(2, 6) . The function of TGase II is less well established. TGase II has been implicated in the activation of several cytokines (7, 8) and in signal transduction(9) . Expression of TGase II has been found in association with apoptosis in several cell types, and a role for TGase II in this process has been suggested(10, 11, 12) . Apoptosis is a genetically controlled process of cell death and is important in the elimination of cells during morphogenesis in embryonic development as well as in many adult tissues(12, 13) . Although the function of TGase II in apoptosis has yet to be elucidated, it has been proposed that TGase II may cross-link cellular proteins, thereby preventing the release of intracellular macromolecules(14) .

RA has been reported to induce TGase II expression in a variety of cell types(4, 15, 16, 17, 18, 19, 20, 21, 22, 23) . In macrophages, the regulation of TGase II expression by RA occurs at the transcriptional level(4) . It is believed that many of the effects of RA on gene expression are mediated by the activation of nuclear retinoid receptors, RARs and/or RXR's (24) . The RAR and RXR gene family each comprises three subtypes named alpha, beta, and (24) . These subtypes are expressed in a developmental stage- and cell type-specific manner(24) , and each may regulate the expression of different genes.

In this study, we examined the retinoid signaling pathways that are involved in the induction of TGase II and apoptosis in rat tracheobronchial epithelial SPOC-1 cells utilizing several novel retinoid receptor-selective agonists and an antagonist. We have also addressed this question through the expression of a truncated RARalpha that acts as a dominant-negative receptor(25) . Our results provide evidence indicating that the induction of TGase II gene expression and apoptosis by RA is mediated through a specific retinoid signaling pathway that involves RARalpha.


EXPERIMENTAL PROCEDURES

Cell Culture

The immortalized, nontumorigenic rat tracheal epithelial cell line SPOC-1 was obtained from Dr. P. Nettesheim (NIEHS). SPOC-1 (passage 7-14) and human lung carcinoma NCI-H460 cells were cultured as described previously(22, 26) . Cells were grown to confluence and then treated with retinoids.

Retinoids

RA was obtained from Hoffmann-La Roche. The RAR-selective retinoid SRI-6751-84 and the RXR-selective retinoid SR11217 were synthesized and characterized as described previously(27, 28) . The RAR-selective retinoid, CD437(29) , was kindly provided by Dr. S. Michel (CIRD/Galderma, Sophia Antipolis, France). The RARalpha-selective retinoid Ro40-6055 (Am580) and the RARalpha-antagonist Ro41-5253 were gifts from Dr. M. Klaus (Hoffmann-La Roche, Basel) (30) . Retinoids were dissolved in dimethyl sulfoxide. The final concentration of dimethyl sulfoxide in the culture medium was 0.5%.

Immunoblot Analysis

Total cellular proteins were analyzed by immunoblot analysis as described previously(18) . Immunoreactivity was determined by a chemiluminescent method using the ECL Western blotting system (Amersham). The mouse monoclonal antibodies Cub7401 against TGase II (31) and B.C1 against TGase I (32) were provided by Drs. P. J. Birckbichler (Oklahoma Medical Research Foundation, Oklahoma City) and S. Thacher (Allergan, Irvine, CA), respectively.

Northern Blot Analysis

Isolation of total and poly(A) RNA and Northern blot analysis were performed as described previously(33) . The TGase II cDNA clone pTG3400 (4) was a gift from Dr. P. Davies (University of Texas, Houston). cDNA probes for RARalpha, -beta, and - and RXRalpha (34, 35, 36) were obtained from Dr. P. Chambon (Strasbourg, France), whereas RXRbeta and - (37) were provided by Drs. D. Mangelsdorf and R. Evans (The Salk Institute, San Diego). A 1.26-kilobase fragment of the chicken glyceraldehyde-3-phosphate dehydrogenase gene (38) was used as a control probe. Following hybridization for 1-2 h at 68 °C using QuickHybry reagent (Stratagene), blots were washed twice with 2 times SSC, 0.1% SDS at room temperature for 15 min. The final wash was with 0.1 times SSC, 0.1% SDS at 65 °C for 30 min. Autoradiography was carried out with Kodak X-Omat AR film at -70 °C using double intensifying screens.

Transient Transfection of SPOC-1 Cells with Constructs Containing RARE-LUC or RXRE-CAT

betaRARE-tk-LUC (39) and (CRBPII)RXRE-tk-CAT (40) constructs were gifts from Drs. V. Giguere (Children's Hospital, Montreal) and D. Mangelsdorf (University of Texas, Dallas), respectively. SPOC-1 cells were grown in 60-mm tissue culture dishes until confluent, and the medium was then changed to 3 ml of serum-free keratinocyte growth medium (Clonetics). The cells were then transfected with 2.5 µg of betaRARE-tk-LUC or co-transfected with 1.5 µg of RXRE-tk-CAT and 1.5 µg of a pSG5 expression vector containing the coding region of RXRalpha (36) using Lipofectin or Lipofectamine reagent (Life Technologies, Inc.). 0.5 µg of beta-actin-LUC or -CAT plasmid DNA was co-transfected to monitor transfection efficiency. Fifteen or five h after transfection, the cells were incubated in fresh growth medium and treated with retinoids for 48 h. Luciferase activity was determined using the luciferase assay system from Promega. CAT activity was assayed as described by Gorman et al.(41) . All experiments were repeated at least twice and performed in triplicate.

Infection of SPOC-1 Cells with Retroviral Vectors

The retroviral vector LRARalpha403SN in which a truncated RARalpha gene is inserted into the retroviral vector LXSN has been described previously (25) . SPOC-1 cells were seeded at the density of 300,000 cells/75 cm^2 flask. The next day, cells were infected with the LXSN or LRARalpha403SN retroviral vector in the presence of 4 µg/ml Polybrene as described previously(25) . After a 5-h incubation, the medium was changed and cells were grown for 24-36 h before G418 (2 mg/ml) was added and the G418-resistant cells were isolated.

DNA Fragmentation Analysis

DNA was isolated as described previously(42) . DNA was electrophoresed in a 1% agarose gel in 1 times TBE buffer (0.05 M Tris base, 0.05 M boric acid, and 1 mM EDTA, pH 8.0) and visualized by ethidium bromide staining.

Electron Microscopic Analysis

Cells were collected, fixed, and embedded in Epon as described(42) . Thin sections (60-90 nm) were stained with 5% uranyl acetate and 2.7% lead citrate and examined in a Phillips ``400'' transmission electron microscope.


RESULTS

Differential Regulation of TGase I and II by RA

Rat tracheal epithelial SPOC-1 cells undergo squamous cell differentiation after reaching confluence and start to express several squamous cell-specific genes including TGase I and cornifin(43) . Treatment of these cultures with RA strongly suppressed the induction of TGase I as revealed by immunoblot analysis using the TGase I-specific antiserum B.C1 (Fig. 1A). In contrast, addition of RA caused an increase in the levels of TGase II protein (Fig. 1A). Suppression of TGase I expression could be observed at concentrations as low as 10M, whereas TGase II was increased at RA concentrations greater than 10M. The level of TGase II was about 20-fold higher in SPOC-1 cells treated with 10M RA as compared to untreated control cells. As shown in Fig. 1B, an increase in TGase II occurred after a lag time of more than 20 h and reached a plateau after 48 h of treatment with 10M RA. The induction of TGase II by RA occurred in logarithmic and confluent cultures (data not shown).


Figure 1: Regulation of TGase I and TGase II by RA in rat tracheal epithelial SPOC-1 cells. A, Confluent cultures were treated for 4 days with RA at the indicated concentrations. Then, total cellular protein was isolated and examined by immunoblot analysis using the TGase I antibody B.C1 or the TGase II mouse monoclonal antibody Cub 7401 as described under ``Experimental Procedures.'' B, confluent cultures were treated with 10M RA for the times indicated and then examined for TGase II by immunoblot analysis.



Expression of RARs and RXRs in SPOC-1 Cells

The induction of TGase II by RA may be directly or indirectly mediated by nuclear retinoid receptors. To examine which retinoid receptors could be involved in this regulation, the expression of RAR and RXR receptors in SPOC-1 cells was investigated. Poly(A) RNAs from RA-, SRI-6751-84-, and dimethyl sulfoxide (control)-treated SPOC-1 cells were subjected to Northern blot analysis. Untreated SPOC-1 cells expressed RARalpha, RAR, and RXRbeta mRNAs (Fig. 2), whereas RARbeta and RXRalpha and - mRNAs were undetectable (data not shown). Treatment of SPOC-1 cells for 4 days with 10M RA or the retinoid SRI-6751-84 increased the level of RAR and RXRbeta mRNAs by about 8- and 7-fold, respectively (Fig. 2). SRI-6751-84 (10M) also induced RXRalpha expression (Fig. 2). RA at 10M did not induce RXRalpha mRNA; however, as shown for TGase II (Fig. 1), 10-10M RA was able to induce RXRalpha (data not shown). In contrast to previous reports on human and rabbit tracheal epithelial cells(44) , RA did not induce RARbeta mRNA in SPOC-1 cells (data not shown).


Figure 2: Expression of RARs and RXRs in SPOC-1 cells. Confluent cultures were treated for 4 days with 10M RA, the RAR-selective retinoid SRI-6751-84 (RAR-Sel.) or vehicle (Control). Poly(A) RNA (5 µg) was fractionated, transferred to a Hybond membrane, and hybridized to P-labeled cDNA probes for mouse RARalpha, -beta, and -, human RXRalpha, -beta, and -, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).



Transactivation of betaRARE-tk-LUC and RXRE-tk-CAT by Receptor-selective Retinoids

To obtain greater insight into which RAR and/or RXR receptor(s) mediate the induction of TGase II, we studied the activity of several retinoids that selectively bind to and activate specific RARs or RXRs. To confirm the activities of these retinoids in SPOC-1 cells, cells were transfected with DNA constructs containing a reporter gene, luciferase (LUC) or chloramphenicol acetyltransferase (CAT), under the control of either an betaRARE or (CRBPII)-RXRE, respectively. The transactivation of the reporter gene was determined after treatment with different retinoid agonists. The RAR-selective retinoid SRI-6751-84, which can bind to and activate all three RAR receptors(27) , was able to induce betaRARE-dependent transactivation of LUC about 27-fold at concentrations 10M through 10M (Fig. 3A). The RARalpha-selective retinoid Ro40-6055 and the RAR-selective retinoid CD437 caused a 19-fold increase in the transactivation of betaRARE-tk-LUC. The RXR-selective retinoid SR11217 (10M), which binds to all three RXR receptors and induces the formation of RXR homodimers(28) , only caused a 6-fold increase in reporter gene activity at 10M. This slight activation of betaRARE-tk-LUC by SR11217 is consistent with the relatively low affinity of RXR homodimers for this response element(45) . In contrast, this RXR-selective retinoid (10M) strongly increased RXRE-dependent CAT activity by about 19-fold, whereas only a 3-fold induction was observed after treatment with RA and SRI-6751-84 at the same concentration (Fig. 3B).


Figure 3: Transactivation of betaRARE-tk-LUC and RXRE-tk-CAT by RA and RAR- and RXR-selective retinoids, SRI-6751-84 (RAR Sel.) and SRI-11217 (RXR Sel.). SPOC-1 cells transfected with betaRARE-tk-LUC (A) and RXRE-tk-CAT (B) were treated with dimethyl sulfoxide or different retinoids at the concentrations indicated. After 48 h of treatment, LUC or CAT activity was measured as described under ``Experimental Procedures.'' beta-Actin-CAT or beta-actin-LUC was included in all transfections to normalize for differences in transfection efficiency. Error bars indicate S.E.



Differential Regulation of TGase II by Receptor-selective Retinoids

Next, we analyzed the effects of these retinoids on TGase II expression. Confluent cultures of SPOC-1 cells were treated for 4 days with these retinoids at different concentrations, and TGase II was examined by immunoblot analysis. As shown in Fig. 4, the RAR-selective retinoid stimulated TGase II at concentrations greater than 10M. SRI-6751-84 was about 100 times more potent than RA (compare row 1 in Fig. 4with row 2 in Fig. 1A). In contrast, the RXR-selective retinoid did not cause any increase in the levels of TGase II protein (Fig. 4). These observations suggest that activation of RXRs is not sufficient for the induction of TGase II. The RARalpha-selective retinoid Ro40-6055 was just as effective in stimulating TGase II expression as RA, whereas the RAR-selective retinoid was unable to induce TGase II (Fig. 4). These results suggest an important role for RARalpha but not RAR in the regulation of TGase II expression.


Figure 4: Induction of TGase II protein by retinoid receptor-selective ligands in SPOC-1 cells. Confluent cultures were treated with SRI-6751-84 (RAR sel.), Ro40-6055 (RAR-alpha sel.), CD437 (RAR- sel.), or SRI-11217 (RXR sel.) at the indicated concentrations for 4 days, cells were then collected and analyzed for the presence of TGase II protein by immunoblot analysis.



The increase in TGase II protein levels by retinoids was related to increased levels of the corresponding mRNA. Northern blot analysis revealed that treatment with the RAR-selective retinoid SRI-6751-84 increased the level of TGase II mRNA in a dose-dependent manner (Fig. 5). The EC for this stimulation was about 1-2 times 10M, which is similar to that for the effect of SRI-6751-84 on TGase II protein levels (compare row 1 in Fig. 4with Fig. 5). However, treatment of SPOC-1 cells with the RXR-selective retinoid SR11217 failed to enhance TGase II mRNA (Fig. 5). These results are consistent with the observations obtained from the immunoblot analysis (compare row 4 in Fig. 4with Fig. 5).


Figure 5: Dose-dependent induction of TGase II mRNA expression by receptor-selective retinoids in SPOC-1 cells. Confluent cells were treated with the RAR-selective retinoid SRI-6751-84 and the RXR-selective retinoid SRI-11217 at the concentrations indicated. After 4 days of treatment, total RNA was isolated, fractionated on 1.0% agarose gel (30 µg/lane), and then transferred to a Hybond membrane. The membrane was successively probed with cDNAs for TGase II and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).



To examine whether the RXR-selective retinoid influences the induction of TGase II by the RAR-selective retinoid, confluent cultures were treated with a suboptimal concentration of SRI-6751-84 (10M) in the presence of increasing concentrations of the RXR-selective retinoid SR11217 for 4 days and then analyzed for TGase II protein. Immunoblot analysis revealed that the RXR-selective retinoid had no effect on the induction of TGase II expression by the RAR-selective retinoid (data not shown).

Effect of an RARalpha Antagonist on Retinoid-induced TGase II Expression

To analyze further the signaling pathway involved in the induction of TGase II by retinoids, the action of the RARalpha antagonist Ro41-5253 was examined. We first determined the effect of Ro41-5253 on the transactivation of betaRARE-tk-LUC. As shown in Fig. 6A, the antagonist was able to inhibit the RARE-dependent transactivation by the RAR-selective retinoid SRI-6751-84 in SPOC-1 cells. We next examined the action of the antagonist on the induction of TGase II. Confluent cultures were treated simultaneously with a suboptimal concentration (10M) of SRI-6751-84 and increasing concentrations of the RARalpha antagonist. After 4 days of treatment, the cells were collected and analyzed for TGase II expression. As shown in Fig. 6B, increasing concentrations of Ro41-5253 inhibited the induction of TGase II by SRI-6751-84. At 10M, Ro41-5253 was able to completely block the induction of TGase II by SRI-6751-84. Ro41-5253 has about a 30-fold lower binding affinity for RARalpha than SRI-6751-84, which may be responsible for the 1000-fold higher concentration required to inhibit TGase II induction. Treatment of SPOC-1 cells with the antagonist alone had no effect on the level of TGase II.


Figure 6: Effect of the RARalpha-selective antagonist Ro41-5253 on the transactivation of betaRARE-tk-LUC and the regulation of TGase II. A, SPOC-1 cells transfected with betaRARE-tk-LUC were treated with a 10M concentration of the RAR-selective retinoid SRI-6751-84 (RAR Sel.) and 2 times 10M Ro41-5253 (Antag.) in the combinations indicated. After 48 h of treatment, cells were collected and the relative LUC activity was determined. B, confluent cells were treated with the RAR-selective ligand (RAR Sel.) in the presence of increasing concentrations of Ro41-5253 (RAR-alpha antag.). After 4 days of treatment, total cellular protein was isolated and examined for TGase II expression by immunoblot analysis.



Effect of Dominant-negative RARalpha403 on TGase II Induction

The observations with the RAR-selective retinoids and the RARalpha-antagonist suggested that a specific RA signaling pathway is involved in the regulation of TGase II gene expression in SPOC-1 cells. To further test this hypothesis, the effect of the truncated RARalpha gene, RARalpha403, on the action of retinoids in SPOC-1 cells was examined. In the human myeloid leukemia cell line HL-60, the LRARalpha403SN exhibits dominant-negative activity and inhibits RA-induced differentiation(25) . To determine whether this construct has dominant-negative activity in SPOC-1 cells, we infected these cells with the amphotropic retroviral vector LXSN (control vector) carrying the neomycin resistance gene and with LRARalpha403SN, which contains the truncated RARalpha cDNA RARalpha403. Subsequently, neomycin-resistant cells were isolated and examined for expression of RARalpha403. Northern blot analysis using poly(A) RNA showed that the LRARalpha403SN-infected SPOC-1 cells expressed high levels of the 4.7-kilobase retroviral transcript containing the RARalpha403 mRNA (Fig. 7A).


Figure 7: Suppression of the retinoid-induced transactivation and TGase II expression in SPOC-1 cells by overexpression of the truncated RARalpha gene RARalpha403. A, RARalpha403 expression in LRARalpha403SN-infected SPOC-1 cells. Poly(A) RNAs from parental SPOC-1 cells and LXSN- and LRARalpha403SN-infected cells were examined by Northern blot analysis using a radiolabeled cDNA probe for RARalpha. B, effect of SRI-6751-84 (RAR-sel.) and Ro40-6055 (RARalpha-sel.) on the transactivation of betaRARE-tk-LUC in parental and LXSN- and LRARalpha403SN-infected SPOC-1 cells. Transfected cells were treated for 24 h with retinoids and then assayed for LUC activity. Results shown are representative for three independent experiments. C, comparison of TGase II induction in parental and LXSN- and LRARalpha403SN-infected SPOC-1 cells. Cells were treated with SRI-6751-84 at indicated concentrations for 4 days and examined for TGase II expression by immunoblot analysis. In this experiment, the induction of TGase II at 10M SRI-6751-84 is higher than that seen in the experiment shown in Fig. 4due to experimental variation.



Next we analyzed the effect of RARalpha403 on the betaRARE-dependent transactivation of CAT (Fig. 7B) and TGase II induction (Fig. 7C) by retinoids. In the parental and LXSN-infected (control) SPOC-1 cells, the RAR-selective (10M) and RARalpha-selective (10M) retinoids induced CAT activity to about the same extent. In the LRARalpha403SN-infected cells, the transactivation by the RAR-selective retinoid was about 50% reduced whereas the transactivation by the RARalpha-selective retinoid was completely abolished. The partial inhibition of the transactivation induced by the RAR-selective retinoid might be due to the fact that the transactivation mediated through RAR is not or is only partially inhibited. As shown in Fig. 7C, SRI-6751-84 induced TGase II equally well in LXSN-infected and parental SPOC-1 cells. However, the induction of TGase II in LRARalpha403SN-infected SPOC-1 cells was considerably less sensitive to SRI-6751-84 than that in LXSN-infected cells (Fig. 7C). In LRARalpha403SN-infected cells, 10M SRI-6751-84 was necessary to induce TGase II. This concentration is about 100-fold higher than that required for the LXSN-infected or the parental SPOC-1 cells.

Correlation between the Induction of TGase II and Apoptosis

Several studies have suggested a role for TGase II in apoptosis(10, 11, 12) . To examine whether the RA-induced TGase II expression in SPOC-1 cells correlated with the induction of apoptosis, we performed DNA fragmentation analysis. As shown in Fig. 8A, treatment of SPOC-1 cells with 10M RA or 10M RAR-selective retinoid SRI-6751-84 for 4 days resulted in the formation of a pattern of fragmented DNA (DNA ladder) typical for cells undergoing apoptosis(12) , whereas no DNA ladder was detected in the samples isolated from cells treated with the RXR-selective retinoid or in control cells. In addition, the induction of fragmented DNA by SRI-6751-84 was abolished by the treatment with the RARalpha antagonist (Fig. 8A). Furthermore, DNA ladders were not observed in the LRARalpha403SN-infected SPOC-1 cells treated with the RAR-selective retinoid (Fig. 8B). The formation of fragmented DNA was first observed after 24 h of treatment with 10M SRI-6751-84 (data not shown). The similarities between the kinetics and the pattern of the induction of TGase II expression and apoptosis by the different retinoids suggest that these two effects are mediated by the same retinoid-signaling pathway and in agreement with the concept that TGase II plays a role in apoptosis.


Figure 8: Electrophoretic analysis of DNA fragmentation in SPOC-1 and human lung carcinoma cells. SPOC-1 cells were treated with 10M RA, 10M RAR- or RXR-selective retinoids or 2 times 10M RARalpha antagonist in the presence or absence of the RAR-selective retinoid for 4 days, and then DNA was isolated and separated by 1% agarose gel electrophoresis. A, parental SPOC-1 cells; B, LRARalpha403SN-infected SPOC-1 cells; C, human lung carcinoma NIH-HUT 460 cells. Cells were treated with RA (10M) or TGFbeta1 (100 pM) for 3 days.



Previously, we showed that RA and TGFbeta1 induce TGase II in several human lung carcinoma cell lines, including adenocarcinoma NCI-H460 cells(22) . Therefore, we wished to investigate whether the induction of TGase II by RA is also accompanied by apoptosis in these cells. As shown in Fig. 8C, treatment of NCI-H460 cells with RA or TGFbeta1 resulted in the formation of fragmented DNA. These findings indicate that the induction of apoptosis by RA is not restricted to SPOC-1 cells and strengthen the correlation between the induction of TGase II and apoptosis.

To obtain further support for the induction of apoptosis by retinoids in SPOC-1 cells, the retinoid-treated and untreated SPOC-1 cells were examined for the presence of apoptotic cells by electron microscopy. Few apoptotic cells (<0.1%) were observed in control cultures and in cultures treated simultaneously with the RARalpha antagonist and the RAR-selective ligand or treated with the RXR-selective retinoid. However, an increased number of apoptotic cells (3-5%) was observed in cultures treated with RA or the RAR-selective retinoid SRI-6751-84. Fig. 9B shows the typical morphology of an apoptotic cell at an early stage of apoptosis in cultures treated with SRI-6751-84. In contrast to the control cell (Fig. 9A), most of the chromatin in the apoptotic cell is aggregated in large compact and segregated granular masses that abut on the nuclear membrane. The cytoplasm is much more condensed than that of the control cell, resulting in crowding of the organelles, which retain their integrity.


Figure 9: Electron micrograph of a normal SPOC-1 cell (A) (magnification times9,000) and an apoptotic cell (B)(magnification times11,200) at an early stage of apoptosis in cultures treated with 10M RAR-selective retinoid for 4 days. Note the condensation and segregation of nuclear chromatin into compact masses abut the nuclear membrane, some of which start to protrude from the membrane.




DISCUSSION

RA has been reported to induce TGase II gene expression in many cell types including several tracheobronchial epithelial cell lines (15, 16, 17, 18, 19, 20, 21, 22) . Although the molecular basis of this regulation has not yet been elucidated, it is likely that retinoid receptors are implicated in this action. In the present study, we examined the signaling pathways that are involved in the retinoid-induced increase of TGase II and apoptosis in rat tracheal epithelial SPOC-1 cells. We demonstrate that these cells express the nuclear retinoid receptors, RARalpha, RAR, and RXRbeta, and, after retinoid treatment, also RXRalpha (Fig. 2). The effects of several receptor-selective retinoids, an RARalpha antagonist and a dominant-negative, truncated RARalpha gene were studied in order to identify the receptor(s) that are involved in these actions. Our studies indicate that the induction of TGase II gene expression in SPOC-1 cells by retinoids is mediated by a specific retinoid signaling pathway that involves the RARalpha receptor. Several lines of evidence support this conclusion. First, the RAR-selective retinoid is a very potent inducer of TGase II (Fig. 4). This retinoid binds specifically to RAR but not to RXR receptors (27) and activates betaRARE-tk-LUC but not RXRE-tk-CAT (Fig. 3)(28) . In contrast, the RXR-selective retinoid which selectively binds to RXRs and induces formation of RXR homodimers (28) was unable to induce TGase II (Fig. 4). However, this retinoid exhibits activity in SPOC-1 cells as indicated by its ability to transactivate RXRE-tk-CAT (Fig. 3B). These observations suggest that activation of RARs plays a role in the induction of TGase II expression by retinoids; however, they do not rule out the involvement of RXRalpha or beta since RARs mediate their activity through a complex with RXRs (46) or other nuclear transcriptional factors(47, 48) . These results suggest that activation of RARs rather than RXRs is a requirement for the induction of TGase II by retinoids in tracheobronchial epithelial cells. This conclusion is in agreement with previous studies showing increased induction of TGase II in rat tracheal 2C5 cells (20) and 3T3 fibroblasts (23) transfected with RAR expression vectors. However, the induction of TGase II in the hematopoietic cell line HL60 cells appears to be dependent on an RXR-dependent pathway since TGase II is induced by RXR-selective retinoids but not by RAR-selective retinoids. (^2)

Second, the induction of TGase II by the RAR-selective retinoid was completely antagonized by the RARalpha antagonist (Fig. 5). The precise mechanism by which the antagonist exerts its effect has not yet been elucidated; however, the antagonist has been shown to compete with RA specifically for binding to RARalpha(30) . Gel-shift experiments indicate that in the presence of the RARalpha-antagonist the RXRbulletRAR complex retains its ability to bind to the RARE(30) . However, the antagonist may be unable to induce the right conformational change in the RARalpha that allows transactivation(30) . The inhibition of the SRI-6751-84-induced transactivation of betaRARE-tk-LUC by this antagonist (Fig. 6A) is in agreement with this concept. The partial inhibition may be due to the fact that the activation mediated by RAR is not inhibited by the antagonist. This is supported by a previous report showing little effect of Ro41-5253 on the betaRARE-dependent transactivation by RAR(30) .

Third, overexpression of the truncated RARalpha gene RARalpha403 in SPOC-1 cells inhibited betaRARE-dependent transactivation and blocked the induction of TGase II by the RAR-selective retinoid (Fig. 7C). The truncated receptor reduced the transactivation of betaRARE-tk-LUC by the RAR-selective retinoid by only 50% but completely suppressed the transactivation by the RARalpha-selective retinoid (Fig. 7B). These results suggests that TGase II induction occurs through RARalpha. Previously, it was reported that the truncated RARalpha403 gene blocks the RA-induced neutrophilic differentiation in HL-60 cells (25) and acts as a dominant-negative receptor. The mechanism by which the truncated RARalpha exerts its dominant-negative action has yet to be elucidated. RARalpha403 is still able to bind RA in in vitro binding assays (49) although with a 12-fold lower binding affinity as the normal RARalpha(49) . It has been demonstrated that the RARs mediate their action as part of a heterodimeric complex with RXR or other transcriptional factors (46, 47, 48) . The product of the LRARalpha403 gene that contains a truncation at its carboxyl terminus is still able to form heterodimers with the RXR receptor(50) . Overexpression of the RARalpha403 may compete for RXR binding with endogenous RARs and inhibit the formation of RARbulletRXR heterodimers and, therefore, the activation of gene transcription. However, it should be noted that RARalpha403 caused only a 50% reduction of the betaRARE-dependent transactivation by the RAR-selective retinoid and did not block the effect of RA on several other genes in SPOC-1 cells. We have found that the effects of retinoids on certain squamous cell-specific genes are not influenced by RARalpha403(43) . These results indicate that the inhibition caused by the RARalpha403 is specific. This specificity may depend on the retinoid signaling pathway that controls the target gene or the type of promoter involved.

Fourth, the RARalpha-selective retinoid Ro40-6055 strongly induced TGase II expression (Fig. 4). This retinoid was about 100 times less active than SRI-6751-84, which is probably related to the reported lower affinity of this RARalpha-selective retinoid to RARalpha(29) . Although the RAR-selective retinoid CD437 was able to transactivate betaRARE-tk-LUC in SPOC-1 cells as effectively as Ro40-6055, it was unable to induce TGase II up to concentrations of 10M. The differential responsiveness of TGase II and betaRARE-tk-LUC to CD437 may be due to differences between the sequence or context of the response elements that determine activation by RAR. Since expression of RARbeta was neither detectable nor inducible by RA in SPOC-1 cells, our results indicate that an RARalpha-dependent rather than an RAR- or RARbeta-dependent signaling pathway is involved in the induction of TGase II expression by RA in SPOC-1 cells.

Although the functions of TGase II have yet to be fully established, recent studies have implicated TGase II in the activation of interleukins (7) and TGFbeta1 (8) and in signal transduction(9) . In several cell types, the expression of TGase II is associated with the induction of apoptosis(10, 11, 12, 14, 51, 52, 53) . However, this correlation is not absolute. Our study demonstrates for the first time that retinoids induce apoptosis in rat and human tracheobronchial epithelial cells as shown by the induction of DNA fragmentation (DNA ladder) (Fig. 8) and by the appearance of cells with typical apoptotic features (Fig. 9). In both SPOC-1 and NCI-H460 cells, this induction is accompanied by an increase in TGase II expression (Fig. 1, Fig. 4, and Fig. 5and (22) ). The pattern by which retinoids induce apoptosis appears to be identical with that for TGase II. Apoptosis was induced by the RAR-selective retinoid and inhibited by the RARalpha antagonist and by the overexpression of the truncated RARalpha gene in a manner similar to TGase II. Future studies have to determine what the role is of TGase II in these cells.

Our results demonstrate that SPOC-1 cells are able to undergo two different processes of irreversible growth arrest, namely squamous differentiation and apoptosis. A schematic representation of the differential regulation of squamous differentiation and apoptosis, and TGase I and TGase II by RA is shown in Fig. 10. RA inhibits squamous cell differentiation as shown by the inhibition of TGase I expression, a squamous cell-specific gene encoding an enzyme that catalyzes the formation of the cross-linked envelope(2, 6) . At the same time, RA is able to induce TGase II and initiate an alternative pathway of cell death, apoptosis (Fig. 1, 4, 5, 8, and 9). These two processes appear to be regulated by different retinoid signaling pathways. This is indicated by the difference in the responsiveness of several squamous-specific genes, such as TGase I (Fig. 1), to the receptor-selective retinoids compared to that of TGase II and apoptosis. In contrast to TGase II and apoptosis, the expression of TGase I and cornifin is regulated by both the RAR- and RXR-selective retinoids and not affected by the truncated RARalpha. (^3)


Figure 10: Schematic representation of the differential regulation of squamous differentiation and apoptosis and TGase I and TGase II by RA in epithelial cells.



In squamous differentiating tissues, a balance exists between the rate of proliferation and differentiation. It has been suggested that retinoids may control the rate at which cells undergo squamous cell differentiation(54) . The regulation of apoptosis may be another mechanism by which retinoids control homeostasis in these tissues.


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.

§
Supported by National Institutes of Health Grant CA-55397.

To whom correspondence and reprint requests should be addressed. Tel.: 919-541-2768; Fax: 919-541-4133; jetten{at}niehs.nih.gov.

(^1)
The abbreviations used are: TGase, transglutaminase; LUC, luciferase; CAT, chloramphenicol acetyltransferase; RA, all-trans-retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; RARE, RAR response element; RXRE, RXR response element; TGF, transforming growth factor.

(^2)
P. J. A. Davies, personal communication.

(^3)
L.-X. Zhang and A. M. Jetten, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Drs. P. Nettesheim for providing the SPOC-1 cells, S. H. Randell for review of this manuscript, and Dr. G. A. Preston for her advice with the analyses of apoptosis. We appreciate the assistance from P. T. Murray and J. L. Horton for EM analysis.


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