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Department of Biochemistry and Molecular Biology, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV 25704
3To whom correspondence should be addressed. E-mail: niles{at}marshall.edu.
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ABSTRACT |
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 TOP ABSTRACT Retinoic acid-induced immediate...Induction of protein kinase...Retinoic acid increases...Role of PKC in...Working model of retinoic...LITERATURE CITED |
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RNA and protein. This gene is not a direct target of RA but appears to be required for the biological effects of RA in B16 melanoma cells. PKC can alter gene transcription via phosphorylation of activator protein (AP)-1. RA increased AP-1 activity in B16 cells but with delayed kinetics compared with activation of PKC by phorbol dibutyrate. Clones stably expressing a dominant negative A-fos gene had reduced AP-1 activity and were less sensitive to RA induction of growth arrest and differentiation. Paradoxically, although inhibition of PKC enzyme activity blocked phorbol dibutyrate-stimulated AP-1 activity, it had no effect on RA-induced AP-1 activity. Further investigation showed that PKC enzyme activity was not required for RA-induced growth inhibition or stimulation of melanin synthesis. These data suggest that PKC either works through a nonenzymatic protein-protein mechanism or may interfere with the enzymatic function of another isozyme of PKC to mediate the actions of RA in B16 melanoma cells.
KEY WORDS: • protein kinase C • activator protein (AP)-1 • melanoma cells • retinoic acid
Melanoma is the most aggressive form of skin cancer. In the United States, its incidence is rapidly increasing, especially in the <35-y-old population (1
). If recognized early, its prompt surgical removal leads to a high cure rate. However, if it reaches the vertical growth phase, melanoma tends to be aggressive and metastasize. In this stage, there is little effective chemotherapy for melanoma. At our laboratory, we have been studying the mechanism by which vitamin A acid [retinoic acid (RA)]4 regulates growth and differentiation of B16 mouse melanoma cells. Unlike many human melanoma cell lines that are resistant to the effects of RA, B16 cells are induced by RA to undergo growth arrest in the G1 phase of the cell cycle and to increase the production of melanin, an indicator of differentiation (2
). By studying in detail how RA induces these changes in mouse B16 cells, we hope to discover ways to overcome the resistance of some human melanomas to treatment with RA.
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Retinoic acid-induced immediate-early genes |
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TOPABSTRACTRetinoic acid-induced immediate...Induction of protein kinase...Retinoic acid increases...Role of PKC in...Working model of retinoic...LITERATURE CITED |
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), we sought to identify the immediate gene targets of RA in B16 melanoma cells. There are several options for profiling gene expression, including DNA arrays, differential displays and serial analysis of gene expression (SAGE). There are advantages and drawbacks to each technology. We chose to use cDNA filter arrays (Atlas arrays; Clontech, Palo Alto, CA). The mouse Atlas arrays used contained 
1200 known genes including RA receptor ß (RARß), which we used as a positive control because RA induces a large increase in its expression in B16 cells (4
). Of the 1200 genes, we found only 4 whose expression was consistently altered by RA (Table 1
). The expression of only two of these four genes was consistently altered in every experiment: RARß and T-box binding protein-2 (Tbx-2).
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). There are at least 21 members of this gene family. The T-box region, which mediates DNA binding, is highly conserved from Drosophila to humans (6
). These transcription factors have been studied mainly in embryonic development, where they seem to be required for tissue-specific development. The expression of Tbx-2 mRNA is increased within 2 h of addition of RA to B16 cells (Fig. 1
). In other data not shown here, we found a degenerate RA response element (RARE) within the first 1 kb of the promoter sequence of the Tbx-2 gene. This element likely mediates at least some of the response to RA. We also found that normal primary cultures of human melanocytes, but not several human melanoma cell lines, express Tbx-2 mRNA.
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). It was discovered as a gene target of the transcription factor MITF (8
). Currently we are developing antibodies against the Tbx-2 protein to further study the role of the transcription factor in RA-induced growth arrest and differentiation. It is interesting to note that all the retinoid-induced immediate-early response genes we have profiled to date are transcription factors. It is likely that these factors in turn target other genes in a cascade of changes in gene expression leading to growth arrest and differentiation of the B16 melanoma cells. |
Induction of protein kinase C by retinoic acid
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TOPABSTRACTRetinoic acid-induced immediate...Induction of protein kinase...Retinoic acid increases...Role of PKC in...Working model of retinoic...LITERATURE CITED |
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mRNA and protein (9
,10
). To investigate the significance of this increase in PKC, we expressed PKC ectopically in B16 cells (Fig. 2
). Clones overexpressing PKC by twofold to fourfold (less than that induced by RA) exhibit a phenotype reminiscent of RA-treated cells (Table 2
). Conversely, down-regulation of PKC by chronic treatment with phorbol dibutyrate antagonizes the ability of RA to inhibit cell growth and stimulate differentiation (11
). Based on these results we conclude that the induction of PKC by RA is necessary for its ability to change the phenotype of B16 mel-anoma cells. There are at least 11 distinct PKC isotypes that differ in their requirement for calcium and diacylglycerol for the activation of their enzymatic activity. In recent experiments we have found that in addition to PKC, B16 cells express PKC
, 
, 
and µ isozymes. RA does not increase the expression of any of the other PKC isotypes; however, RA did decrease the expression of PKC and PKCµ. (unpublished data).
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Retinoic acid increases activator protein-1 transcriptional activity |
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TOPABSTRACTRetinoic acid-induced immediate...Induction of protein kinase...Retinoic acid increases...Role of PKC in...Working model of retinoic...LITERATURE CITED |
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, we determined whether AP-1 activity was also increased. Using transient transfection of B16 cells with an AP-1 luciferase reporter gene, and more recently using B16 clones stably expressing an AP-1-luciferase reporter gene, we have found that retinoic acid induces a twofold to fourfold increase in AP-1 transcriptional activity (12
). However, the kinetics of RA-induced AP-1 activity are quite different from that induced by phorbol dibutyrate (Fig. 3
). In the case of PKC activation by phorbol dibutyrate, AP-1 activity rapidly increases and then decreases to baseline levels by 24 h of treatment. In contrast, in cells treated with retinoic acid, AP-1 activity does not increase until 24 h, reaches a maximum by 48–56 h and then remains at this level for as long as we have treated cells with retinoic acid (72 h).
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).
To determine the importance of RA-induced AP-1 activity, we established clones of B16 melanoma cells that stably express a dominant negative version of c-fos termed A-fos (13
). These clones have markedly reduced expression of AP-1 in response to treatment with either phorbol dibutyrate or RA. The phenotype of the clones is shown in Table 3
. Surprisingly, the growth rate of A-fos-expressing clones that were not treated with RA was not very different from that of wild-type cells, indicating that these cells have growth factor-stimulated pathways that do not involve AP-1. However, A-fos-expressing clones exhibited an attenuation in the ability of RA to inhibit anchorage-dependent and -independent growth. In addition, there was a complete loss of the ability of RA to increase melanin production in A-fos-expressing B16 cells.
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). In contrast, the down-regulation of PKC induced by long-term treatment of B16 cells with phorbol dibutyrate decreased RA-induced AP-1 activity. Cumulatively, these data suggest that RA-induced AP-1 activity is required for at least some of the phenotypic changes induced by vitamin A and that alteration of PKC protein, but not PKC enzyme activity, is a part of the pathway by which RA increases AP-1 activity. |
Role of PKC in RA-induced melanoma growth arrest and differentiation |
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TOPABSTRACTRetinoic acid-induced immediate...Induction of protein kinase...Retinoic acid increases...Role of PKC in...Working model of retinoic...LITERATURE CITED |
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summarizes the effect of the PKC enzyme inhibitor bisindolylmaleimide on retinoid-induced growth inhibition and melanin production. Inhibition of PKC enzyme activity did not alter the ability of RA to decrease anchorage-dependent and -independent growth nor block its ability to stimulate melanin production. Indeed, decreasing PKC enzyme activity by itself inhibited cell growth and increased melanin production.
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protein while the inhibition of PKC enzyme activity did not block retinoid action was addressed by ectopically expressing two different mutant forms of PKC in B16 cells. The two PKC mutants used in this study are illustrated in Figure 4
. One has a mutation in the ATP-binding site and therefore lacks catalytic activity. The second mutant has two amino acid substitutions in the pseudosubstrate domain of the protein. Normally, this would result in a constitutively active enzyme. However, this construct also has a mutation in the ATP-binding site. Therefore the protein that is encoded by this construct is predicted to have an "open" conformation but to lack enzymatic activity. We used these two different constructs to determine whether some protein-protein interactions may be dependent on the "open" conformation of PKC. The phenotype of several B16 clones expressing these mutated PKC proteins is compared with wild-type cells or B16 clones not expressing these mutant enzymes in Table 5
. The correlation between the decreased PKC enzyme activity in these clones and their altered phenotype is noteworthy. The only retinoid-induced change in wild-type cells that was not mimicked in B16 clones expressing the mutant PKC isozymes was the increase in melanin production.
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protein may possess nonenzymatic function(s) that are necessary for the production of some of the phenotypic changes seen in these cells. Other investigators have reported that PKC has nonenzymatic functions (14
,15
). An alternative explanation of the results is that inhibition of PKC enzyme activity is responsible for the observed phenotype of these clones; this would agree with some of the results obtained from the use of a PKC enzyme inhibitor (Table 4)
. However, the enzyme inhibitor increased melanin production and decreased AP-1 transcriptional activity, actions that are opposite to those observed in the clones expressing PKC mutant proteins. As mentioned earlier, we have found that in addition to PKC, B16 cells express PKC, , and µ. RA decreases the expression of PKC and µ. Perhaps these opposite actions of RA on different PKC isozymes can explain the paradox of the results described earlier. Currently we are using PKC isozyme-specific antisense constructs to probe the functions of various isoforms that are affected in RA-treated B16 cells. |
Working model of retinoic acid induction of B16 melanoma growth arrest and differentiation |
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TOPABSTRACTRetinoic acid-induced immediate...Induction of protein kinase...Retinoic acid increases...Role of PKC in...Working model of retinoic...LITERATURE CITED |
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depicts a schematic of our current understanding of the pathways involved in the mechanism by which RA inhibits growth and stimulates the differentiation of B16 mouse melanoma cells. We have not studied how RA arrives in the nucleus to bind to the nuclear receptors, but recent evidence from other systems indicates that cellular RA-binding protein (CRABP)-II might be involved (16
). At least two of the primary gene targets are Tbx-2 and RARß. Two other genes, namely, c-fos (verified by Northern and Western blotting, unpublished results) and Hox-2a (yet to be verified), are also likely to be directly regulated by RA. It is interesting that all of these target genes are transcription factors. Thus it is likely that there is a secondary wave of altered gene expression induced by these factors. PKC is probably one of these secondary target genes because its mRNA and protein are induced by sixfold to eightfold in retinoid-treated cells and the mRNA increase is blocked by inhibitors of protein synthesis. In addition to transcriptional regulation, there is significant posttranscriptional regulation of PKC mRNA in RA-treated B16 cells. The exact role of the quantitative increase in PKC is a paradox. On the one hand, overexpression of PKC in B16 cells results in a phenotype resembling that induced by RA treatment. Also down-regulation of PKC antagonizes the action of RA. However, inhibition of PKC enzyme activity also mimics the phenotype of RA treatment: that is, decreased anchorage-dependent and -independent growth and increased melanin production. Part of these conflicting results might be due to altered activity of other PKC isotypes, but evidence suggests that nonenzymatic functions of PKC might also play a role. Further down one of the pathways is the stimulation of AP-1 transcriptional activity, which appears to be necessary for maximal effect of RA. The mechanism by which RA increases AP-1 activity is still unknown, but it does not require PKC enzyme activity. Because there is a time lag of about 24 h before increased AP-1 activity can be detected in RA-treated B16 cells, it is likely that increased expression of one or more gene products contributes to this increased activity. Because AP-1 is a transcription factor complex, it is likely that its target genes contribute to the final phenotype of growth arrest and differentiation. Our research has focused on early events in the RA-induced pathway of melanoma growth arrest and differentiation and we do not yet know the cause of the growth arrest in the G1 phase of the cell cycle. Based on studies on retinoid-induced growth arrest in other cell types, it is likely to be due, ultimately, to changes in cell cycle regulatory proteins such a decrease in G1 phase cyclins and or an increase in cyclin-dependent kinase inhibitors (17
,18
). We are hopeful that the information we have obtained through the dissection of the pathways by which RA affects the growth and differentiation of B16 melanoma cells will provide some insight into why many human melanomas are relatively resistant to retinoid-induced growth arrest and differentiation.
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FOOTNOTES |
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2 Supported in part by National Institutes of Health grant RO1-CA59530 and Ameican Institute for Cancer Research grant 99B041.
4 Abbreviations used: AP-1, activator protein-1; PKC, protein kinase C; RA, retinoic acid; Tbx-2, T-box binding protein-2.
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LITERATURE CITED |
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TOPABSTRACTRetinoic acid-induced immediate...Induction of protein kinase...Retinoic acid increases...Role of PKC in...Working model of retinoic...LITERATURE CITED |
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11. Gruber, J., Ohno, S. & Niles, R. M. (1992) Increased expression of PKC plays a key role in retinoic acid-induced melanoma differentiation. J. Biol. Chem. 267:13356-13360.
12. Desai, S. & Niles, R. M. (1997) Characterization of retinoic acid-induced AP-1 activity in B16 melanoma cells. J. Biol. Chem. 272:12809-12815.
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14. Singer, W. D., Brown, H. A., Jiang, X. & Sternweis, P. C. (1996) Regulation of phospholipase D by protein kinase C is synergistic with ADP-ribosylation factor and independent of protein kinase activity. J. Biol. Chem. 27:4504-4510.
15. Diaz-Meco, M. T., Municio, M. M., Sanchez, P., Lozano, J. & Moscat, J. (1996) Lambda-interacting protein, a novel protein that specifically interacts with the zinc finger domain of the atypical protein kinase C isotype lambda/iota and stimulates its kinase activity in vitro and in vivo. Mol. Cell Biol. 16:105-114.[Abstract]
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17. Dimberg, A., Bahram, F., Karlberg, I., Larsson, L. G., Nilsson, K. & Oberg, F. (2002) Retinoic acid-induced cell cycle arrest of human myeloid cell lines is associated with sequential down-regulation of c-Myc and cyclin E and posttranscriptional up-regulation of p27(Kip1). Blood 99:2199-2206.
18. Zhang, D., Vuocolo, S., Masciullo, V., Sava, T., Giordano, A., Soprano, D. R. & Soprano, K. J. (2001) Cell cycle genes as targets of retinoid induced ovarian tumor cell growth suppression. Oncogene 20:7935-7944.[Medline]
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, both of which are constitutively expressed in these cells. Two known primary gene targets of RAR/RXR in these cells are RARß and Tbx-2. The gene targets of Tbx-2 are unknown, but may ultimately contribute to the RA-induced AP-1 activity. It is postulated that one of the gene targets of RARß is PKC