Retinoic acid and 4-hydroxyphenylretinamide induce growth inhibition and tissue transglutaminase through different signal transduction pathways in mouse fibroblasts (NIH 3T3 cells)
Valeria Giandomenico,
Fausto Andreola,
Maria Luisa Rodriguez de la Concepcion,
Steven J. Collins1 and
Luigi M. De Luca2
Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255 and
1 Fred Hutchinson Cancer Research Center, Seattle, WA 98104, USA
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Abstract
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4-Hydroxyphenylretinamide (4-HPR) is a synthetic retinoid with minimal toxicity and favorable pharmacokinetics during long-term administration to patients in clinical trials. Since 4-HPR binds poorly to the retinoic acid receptors, the issue of whether 4-HPR exerts its biological actions via classical retinoid receptor pathways remains to be resolved. We have previously reported that stable expression of a truncated retinoic acid receptor
, RAR
403, transduced in NIH 3T3 cells by a retroviral vector, rendered the cells resistant to retinoic acid for growth inhibition and induction of tissue transglutaminase (TGase II). Here, we report that stable expression of the dominant negative construct RAR
403 fails to blunt growth inhibition and TGase II induction by 4-HPR, a potent chemopreventive retinoid, in the same cells. These data show that retinoic acid receptors do not mediate either growth inhibition or induction of TGase II activity by 4-HPR in mouse fibroblast cells.
Abbreviations: 4-HPR, 4-hydroxyphenylretinamide; RA, all-trans-retinoic acid; RAR, retinoic acid receptor; TGase II, tissue transglutaminase.
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Introduction
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All-trans-retinoic acid (RA) induces cell growth inhibition, apoptosis and tissue transglutaminase (TGase II) in different cell types (14). We have previously reported that retinoic acid receptors mediate the growth inhibitory action and the induction of TGase II activity by RA in mouse fibroblasts (5,6). The synthetic retinoid 4-hydroxyphenylretinamide (4-HPR) inhibits cell proliferation and induces apoptosis even in RA-resistant cell lines (7,8). 4-HPR has been suggested to function through retinoic acid receptor (RAR)
. However, its binding constant for this receptor is relatively low compared with that of RA (9). Others have suggested that 4-HPR is not a `true' retinoid (9,10), but proof that 4-HPR action does not involve the retinoid receptors is still lacking.
Tissue TGase II is a ubiquitous cytoplasmic enzyme involved in cell adhesion (11) and was recently recognized as an important enzyme in cell growth inhibition and apoptosis in several systems (1,2). TGase II is a RA target gene and the presence of a versatile retinoid response element has been shown in the promoter region of the mouse gene (12). The involvement in cell growth inhibition and the presence of a retinoid response element in its promoter made TGase II the elective target gene for our study.
We have shown that 4-HPR induces growth inhibition and TGase II activity in several cell types, including NIH 3T3 cells (13). In this study we asked whether 4-HPR uses the RAR-dependent signaling transduction pathway to induce these phenomena. We addressed this question in NIH 3T3 cells expressing RAR
403, a truncated form of the RAR
with dominant negative activity over the function of all RARs (14). We introduced RAR
403 into NIH 3T3 cells by retrovirus infection with a construct, LXRAR
403SN, which contains a truncated RAR
gene inserted into the retroviral vector LXSN (14). Northern blot analysis detected the expression of the typical 4.7 kb retroviral transcripts containing the RAR
403 mRNA (Figure 1
). Expression of the endogenous RAR
transcripts at 3.7 and 2.6 kb was also observed (Figure 1
). The dominant negative activity of the mutated receptor was confirmed in LXRAR
403SN-NIH 3T3 cells by transient reporter assays, as shown previously (15; data not shown).
The effects of RA and 4-HPR on the proliferation of control LXSN-NIH 3T3 and LXRAR
403SN-NIH 3T3 cells were determined using Cell Titer 96AQueous One Solution Cell Proliferation Assay kit according to the manufacturer's manual (Promega). The effects of RA and 4-HPR over 72 h on cell growth are shown in Figure 2
. RA inhibited growth of control LXSN-NIH 3T3 cells, but failed to inhibit LXRAR
403SN-NIH 3T3 cell proliferation. In sharp contrast, the inhibitory effect of 4-HPR on cell proliferation was observed in both control LXSN- and LXRAR
403SN-NIH 3T3 cells. 4-HPR inhibited NIH 3T3 cells as well (data not shown).
Next, we measured the effect of RA and 4-HPR on TGase II enzyme activity. Cell lysates were assayed for TGase II by measuring [2,3-3H(N)]putrescine incorporation into dimethyl casein, according to Lichti and Yuspa (16). In agreement with our previous results (6), RA strongly induced TGase II activity (Figure 3A
). Moreover, expression of RAR
403 strongly inhibited induction of TGase II by RA (Figure 3A
), confirming the involvement of RARs in this pathway. TGase II activity was slightly stimulated by 1 µM 4-HPR. However, Figure 3B
shows that 10 µM 4-HPR induced TGase II activity by >4-fold in both control LXSN-NIH 3T3 and infected LXRAR
403SN-NIH 3T3 cells. A similar induction was obtained in NIH 3T3 cells (13).
To address whether the observed induction of TGase II activity corresponds to an increase in TGase II RNA levels, we performed northern blot analysis of poly(A)+ mRNA. TGase II mRNA was increased after 48 h of RA treatment in LXSN-NIH 3T3 cells. LXRAR
403SN-NIH 3T3 cells were, however, unresponsive (Figure 4
). Interestingly, there was no increase in TGase II mRNA in either LXSN-NIH 3T3 or LXRAR
403SN-NIH 3T3 cells after treatment with 4-HPR (Figure 4
).

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Fig. 4. Effects of RA and 4-HPR on TGase II mRNA expression. Cells were treated with DMSO, RA (106 M) or 4-HPR (106 and 105 M) for 48 h. RA greatly increased TGase II mRNA in LXSN-NIH 3T3 cells, but failed to increase transcription of TGase II in LXRAR 403SN-NIH 3T3 cells. The 3.348 kb fragment of mouse TGase II was excised from the expression plasmid pGEM3Z. Human GAPDH was used as a loading control. 4-HPR failed to increase TGase II mRNA level in both cell lines. GAPDH was used as a quantitative loading control. Any impression of up-regulation in LXSN-NIH 3T3 by 4-HPR is due to unequal loading. The experiment was repeated twice with similar results in two different RNA preparations.
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Therefore, TGase II is mainly regulated at the level of mRNA by RA through an RAR-dependent signaling pathway, as already shown (6,12,17). However, we still don't know whether this control is due to increased transcription or increased stability of TGase II mRNA.
In contrast, 4-HPR acts through an RAR-independent molecular mechanism. This retinoid might regulate TGase II enzyme activity through a post-transcriptional control. This same mechanism might be responsible for the residual induction of TGase II by RA in LXRAR
403SN cells (Figure 3A
).
In conclusion, this work demonstrates for the first time that RARs are not the mediators of 4-HPR-induced inhibition of cell growth and stimulation of TGase II enzymatic activity in mouse fibroblast cells.
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Acknowledgments
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We thank Dr Reuben Lotan for the mTGase II cDNA and Drs Keiko Ozato and Bruno Lefebvre for the mRAR
cDNA and the construct containing ß-RAREtkluciferase.
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Notes
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2 To whom correspondence should be addressed at: Building 37 Room 3A/17, 37 Convent Drive, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA Email: luigi_de_luca{at}nih.gov 
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Received November 20, 1998;
revised February 5, 1999;
accepted March 1, 1999.