Several cell lines derived from in vivo tumors have demonstrated resistance to the growth inhibitory influences of 1,25(OH)2
). It is not uncommon to observe the spontaneous development of resistance to a growth inhibitory substance in human cancer. The occurrence of 1,25(OH)2
resistance reported in this study is not associated with a genetic alteration of the VDR (17
). In addition, Western analyses showed that hRXRα is expressed at similar levels in HPK1A and HPK1Aras
cells, suggesting that partial resistance to 1,25(OH)2
cells does not arise from increased degradation of hRXRα. Instead, this study indicates that transcriptional activation by the VDR is attenuated through phosphorylation of the heterodimeric partner hRXRα by MAP kinase. This posttranslational modification is mediated through overexpression of the ras
oncoprotein, which induces the Ras-Raf-MAP kinase cascade in HPK1Aras
cells and stimulates phosphorylation of hRXRα on ser260.
The resistance phenomenon observed in HPK1Aras
cells not only affects cell growth, but also affects endogenous genes known to be important in the control of cell growth and differentiation (13
). In particular, the observed resistance to parathyroid hormone–related peptide (PTHrP) expression by HPK1Aras
cells detected previously (25
) is especially relevant because PTHrP was found to be antiproliferative (26
) and prodifferentiative (26
) in HPK1A cells.
Although our present study defines a specific cellular model, the potential ramifications of these findings are extensive. 1,25(OH)2
inhibits the growth of tumors derived from a variety of tissues (27
). Given the high frequency of ras
activation in human cancers, phosphorylation of hRXRα may be a common mechanism by which cancer cells escape the growth inhibitory effects of the endogenously produced hormone 1,25(OH)2
. Moreover, hRXRα functions in a wide variety of transcriptional responses through its heterodimerization with several nuclear receptors in addition to the VDR, including thyroid hormone receptors (TRs), retinoic acid receptors (RARs), peroxisome proliferator–activated receptors (PPARs), and several orphan receptors (18
). Signaling through heterodimeric partners of RXRs other than the VDR can modulate cellular growth and differentiation. For example, PPARα promotes differentiation of adipocytes (28
) and has recently been shown to inhibit growth of colon cancer cell lines (29
). Consequently, disruption of signal transduction by members of this superfamily of receptors could have multiple consequences for the control of cell growth and differentiation.
Through studies with the RXR-specific ligand LG1069, we have demonstrated that this MAP kinase phosphorylation of hRXRα in ras
-transformed keratinocytes affects another signaling pathway that relies on the RXR. The ras
-transformed cells are not only resistant to the growth inhibitory action of 1,25(OH)2
but are also resistant to the growth inhibitory action of LG1069. Furthermore, we have shown previously in gel retardation assays a similar phenomenon in thyroid hormone signaling; the RXR antibody also fails to shift TR/hRXRα heterodimers formed on thyroid hormone response elements (TREs) (17
Targeting hRXRα confers a growth advantage to cells that have been transformed by ras by affecting several signaling pathways that depend on RXR. Because 1,25(OH)2D3 is a powerful inhibitor of cell growth, it is beneficial for a cancer cell to resist this inhibitory pathway. In addition, by affecting the common link among many nuclear receptor pathways, cells transformed by ras are able to influence signal transduction in a much broader sense. Although this study focused on 1,25(OH)2D3 action, we have demonstrated that phosphorylation of hRXRα on ser260 also affects RXR/RXR signaling, indicating that this is a common mechanism used by ras-transformed keratinocytes to interfere with hormone signaling.
Gel retardation assays performed in the presence of specific antibodies raise the possibility that phosphorylation of hRXRα on ser260 induces a conformational change within the receptor that causes attenuation of ligand-dependent transactivation. An anti-RXR antibody with an epitope spanning ser260 was unable to recognize and therefore supershift the VDR/RXR complex from the ras
-transformed keratinocytes (Figure a), whereas an anti-RXR antibody possessing an epitope in the NH2
-terminal domain of hRXRα remote from ser260 was able to recognize and therefore inhibit the formation of a VDR/RXR complex on the 32
P-labeled VDRE (Figure b). Phosphorylation of ser260 occurs at a critical site close to regions of potential coactivator interaction with the RXR (30
). Taken together, these results indicate that a conformational change within the LBD of hRXRα probably occurs following phosphorylation of ser260.
Indeed, several adaptor proteins that can act as coactivators for nuclear receptors have been identified recently (32
), including the coactivator SRC-1 (steroid receptor coactivator-1), which can interact with the LBD of steroid receptors to increase ligand-dependent transcriptional activation (33
). We cannot exclude the possibility that phosphorylation of ser260 of hRXRα may disrupt the interaction of this heterodimeric partner with SRC-1 or other coactivators and thus attenuate ligand-dependent transactivation. Irrespective of the exact consequences of RXR phosphorylation on RXR conformation, our study has identified a new target of ras
activation that may play a critical role in malignant transformation.