Mechanisms of nuclear vitamin D receptor resistance in Harvey-ras-transfected cells

Abstract

The hormone 1,25 dihydroxyvitamin D (1,25(OH)(2)D) binds to the nuclear vitamin D receptor (nVDR), which heterodimerizes with retinoid X receptor alpha (RXRalpha), and this complex interacts with specific response elements [vitamin D response elements (VDREs)] to regulate gene transcription. Previous results show a significant reduction in 1,25(OH)(2)D-induced nVDR transcriptional activity in fibroblast (C3H10T1/2) cells transfected with the Harvey ras gene (ras cells) compared with parental cells. The purpose of this study was to investigate the mechanisms by which the H-ras gene interferes with nVDR transcriptional activity. Similar to the ras cells, transcriptional activity of the nVDR was reduced following induction of the H-ras gene for 9 days. The ras cells expressed similar protein levels of RXRalpha with the parent cells, and overexpression of the wild-type RXRalpha plasmid did not restore 1,25(OH)(2)D-mediated nVDR activity in ras cells. Inhibiting activation of extracellular signal-regulated kinase (ERK1/2) had no effect on nVDR activity in ras cells. Furthermore, the binding of nVDR to VDREs was reduced in 1,25(OH)(2)D-treated ras cells. In addition, neither treatment of ras cells with an inhibitor (ketoconazole) of the 1,25(OH)(2)D degradative enzyme, 24-hydroxylase, nor the protein kinase C inhibitors, bisindoylmaleimide I and Gö 6976, had an effect on nVDR activity. In contrast, inhibition of phosphatidylinositol 3-kinase (PI3K) with LY294002 resulted in a 1.6-fold significant increase in the nVDR activity in the ras cells. Taken together, these results indicate that PI3K may, at least in part, mediate the suppression of the 1,25(OH)(2)D regulation of nVDR transcriptional activity by the H-ras gene, leading to reduced ability to associate with response elements.

Fig. 1

Modulation of nVDR-mediated transcriptional regulation by H-ras oncogene. The presence of the H-ras oncogene reduces transcriptional activity of the nVDR. The H-ras oncogene leads to increased activity of PKC, PI3K and ERK1/2 which may subsequently regulate the activity of the nVDR or its heterodimer partner RXR. In addition, 1,25(OH)₂D is degraded through the activity of the 24-hydroxylase, which may reduce the nVDR activity by reducing cellular ligand concentration. The liganded nVDR with the RXR interacts with VDREs in the DNA to induce CYP24 expression, and the H-ras oncogene may impact the ability of this complex to interact with the VDRE.

Fig. 2

Induction of Harvey-ras gene in pMTrasneo 13 cells following zinc chloride treatment. (A) A representative Northern blot showing Harvey-ras mRNA in pMTrasneo13 cells after 24 and 48 h of exposure to 50 μM zinc chloride. The left two lanes show Harvey-ras mRNA levels from rasneo11A cells (ras) and C3H10T1/2 cells (10T1/2) which serve as controls. (B) Results of immunoprecipitation of radiolabeled methionine-labeled cells showing increased p21^RAS protein in pMTrasneo13 cells following treatment with 50 μM zinc chloride for indicated times. The left two lanes contain proteins isolated from C3H10T1/2 cells (10T1/2) following zinc chloride treatment for 0 and 48 h. (C) Phenotype of pMTrasneo13 cells without (upper left panel) and with (upper right panel) addition of zinc chloride to the media for 3 weeks. Phenotype of pMTrasneo13 cells at high density without (lower left panel) and with (lower right panel) addition of zinc chloride to the media for 3 weeks. (D) nVDR transcriptional activity in pMTrasneo13 cells. pMTrasneo13 cells were pretreated with 50 μM zinc chloride for 7 days, transiently co-transfected with the CYP24 luciferase and Renilla luciferase control plasmids for 24 h, and treated with 100 nM 1,25(OH)₂D or 0.1% ethanol vehicle control for 24 h. CYP24 luciferase activity was assayed and normalized for transfection efficiency via Renilla luciferase activity. Results are expressed as relative luciferase units (sample/vehicle). Values are means±S.E.M. (n=3); asterisk indicates P<.001 between groups.

Fig. 3

Role of RXRα in nVDR transcriptional activity in stably Harvey-ras transfected cells. (A) RXRα protein expression in C3H10T1/2 and rasneo11A cells. C3H10T1/2 and rasneo11A cells were treated with ethanol vehicle or 100 nM 1,25(OH)₂D for 24 h and probed with RXRα antibody. The upper panel is a representative autoradiograph of RXRα and the lower panel is the actin control of a Western blot of three separate cell preparations with RXRα C3H10T1/2 (10T1/2), rasneo11A and control protein cell lysates. (B) Histogram representative of the quantification of RXRα protein expression by Western blot analysis (RXRα/actin control). Results are expressed as relative optical density. Values are means±S.E.M. (n=3). (C) nVDR transcriptional activity in rasneo11A cells following transient transfection with empty plasmid, RXRα (A260S) mutant plasmid (RXR-SER²⁶⁰) or overexpression of wild-type RXRα (RXR^WT). Rasneo11A cells were transiently co-transfected with the CYP24 luciferase plasmid and Renilla luciferase control plasmids for 24 h. Each of the three groups was treated with 0.1% ethanol vehicle control or 100 nM 1,25(OH)₂D for 24 h. CYP24 luciferase activity was assayed and normalized for transfection efficiency via Renilla luciferase activity. Results are expressed as relative luciferase units (sample/vehicle). Values are means±S.E.M. (n=3). There was no significant effect between groups as determined by one-way ANOVA. (D) Effect of MAPKK/MEK inhibition of nVDR transcriptional activity. Rasneo11A cells were transiently co-transfected with the CYP24 luciferase and Renilla luciferase control plasmids for 24 h, pre-treated with 50 μM PD98059 for 1 h and co-treated with 0.1% DMSO and 0.1% ethanol vehicle control or 100 nM 1,25(OH)₂D for 24 h. CYP24 luciferase activity was assayed and normalized for transfection efficiency via Renilla luciferase activity. Results are expressed as relative luciferase units (sample/vehicle). Values are means±S.E.M. (n=3); there was o significant effect between groups.

Fig. 4

Electromobility shift assay in 1,25(OH)₂D-treated cells. A nVDR/RXR oligo probe was labeled with ³²P and incubated with 100 nM 1,25 (OH)₂D (1,25D) or ethanol vehicle (V) control-treated nuclear extracts from C3H10T1/2 and rasneo11A cells. The nVDR/DNA complex is indicated. Lanes are electronically arranged for consistency in presentation.

Fig. 5

Effect of PKC in C3H10T1/2 and rasneo11A cells on nVDR transcriptional activity. C3H10T1/2 and rasneo11A cells were transiently co-transfected with the CYP24 luciferase and Renilla luciferase control plasmids for 24 h, pretreated with 2 μM bisindoylmaleimide I or 10 nM Gö 6976 for 6 h and treated with 0.1% DMSO and 0.1% ethanol vehicle control or 100 nM 1,25(OH)₂D for 18 h. CYP24 luciferase activity was assayed and normalized for transfection efficiency via Renilla luciferase activity. Results are expressed as relative luciferase units (sample/vehicle). Values are means of one representative experiment for each cell type± S.E.M. (n=3). Bars with different letters indicate significant differences in treatment within cell type (P<.05).

Fig. 6

Role of PI3K in 1,25(OH)₂D-induced nVDR transcriptional activity. Inhibition of PI3K with LY294002. C3H10T1/2 and rasneo11A cells were transiently co-transfected with the CYP24 luciferase and Renilla luciferase control plasmids for 24 h, pretreated with 50 μM LY294002 for 6 h and treated with 0.1% DMSO and 0.1% ethanol vehicle control or 100 nM 1,25 (OH)₂D for 18 h. CYP24 luciferase activity was assayed and normalized for transfection efficiency via Renilla luciferase activity. Results are expressed as relative luciferase units (sample/vehicle). Values are means±S.E.M. For C3H10T1/2 cells, two experiments (n=5). For rasneo11A cells, three experiments (n=8); asterisk indicates significant differences between treatments within group (P=.0003).