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Vitamin D interferes with glucocorticoid responsiveness in human peripheral blood mononuclear target cells

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Abstract

Glucocorticoids (GCs) are widely used in the treatment of inflammatory and autoimmune diseases; however, patients are often resistant to GC effects. Current studies indicate that vitamin D reduces the risk or modifies the course of autoimmune diseases posing vitamin D supplementation as a prevention or therapeutic option. Herein, we investigated whether vitamin D can modify the response to GCs at the molecular level. To this end, peripheral blood mononuclear cells (PBMCs) were isolated from healthy vitamin D-deficient women and incubated with either the active metabolite 1,25(OH)2D3 (VitD) for 11 days or dexamethasone (Dex) for the last 2 days in the presence or absence of VitD. Ex vivo GC sensitivity was assessed by the expression of the glucocorticoid receptor (GR) responsive gene GILZ with RT-PCR. Long-term incubation of PBMCs with VitD significantly decreased the Dex-induced augmentation of GILZ expression. Since the intracellular concentration of GR and the GR nuclear translocation are critical determinants of GC sensitivity, we next evaluated the effect of VitD on these factors. RT-PCR and western-blot analysis revealed that VitD reduced the expression of GR. This effect was abolished by the HDAC-specific inhibitor trichostatin A, implying that HDAC was implicated in this effect. Moreover, NCoR1 mRNA was significantly decreased upon treatment with VitD either alone or as pre-treatment to Dex, suggesting that a possible increase in expression of this co-repressor was not involved. In addition, immunofluorescence analysis showed that VitD hindered the Dex-induced GRα nuclear translocation, an effect verified by subcellular fractionation and western-blot experiments. To further explore the underpinning mechanism, we examined the potential of VitD to: (1) strengthen the FK506-binding protein 5 (FKBP5) negative feedback loop and (2) modify the phosphorylation status of GR. Remarkably, VitD decreased FKBP5 expression and decreased phosphorylation at Ser211, while enhancing phosphorylation of GR at Ser203. Overall, VitD decreases the ex vivo GC sensitivity and this effect is, at least in part, attributed both to decrease of GR expression owing to a mechanism that engages HDAC and inhibition of GR translocation to nucleus via differential modulation of the phosphorylation state of GR. Our study provides, for the first time, evidence that long-term action of VitD induces GC resistance in PBMCs from healthy volunteers and offers a possible mechanistic basis for VitD-triggered attenuation of GC effects.

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Abbreviations

CBG:

Corticosteroid-binding globulin

ERK:

Extracellular signal-regulated kinase

FBS:

Fetal bovine serum

FKBP5:

FK506 binding protein 5

GCs:

Glucocorticoids

GILZ:

Glucocorticoid-induced leucine zipper

GREs:

Glucocorticoid response elements

HDAC3:

Histone deacetylase 3

HSPs:

Heat shock proteins

IF:

Immunofluorescence

MAPK:

Mitogen-activated protein kinase

MKP-5:

Mitogen-activated protein kinas phosphatase 5

PBMCs:

Peripheral blood mononuclear cells

PP5:

Protein phosphatase 5

TSA:

Trichostatin A

VDR:

Vitamin D receptor

VitD:

Vitamin D

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Acknowledgments

We thank Dr. J. Cidlowski (Laboratory of Signal Transduction and Laboratory of Integrative Bioinformatics, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Triangle Park, NC, USA) for providing the anti-phospho-GR-S203 antibody and Dr. G. Dalagiorgou (Department of Biological Chemistry, Medical School, National and Kapodistrian University of Athens) for help with immunofluorescence. This work was supported by a grant from Unipharma (Grant Code 70/3/13029).

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    Authors and Affiliations

    1. Department of Biological Chemistry, Medical School, National and Kapodistrian University of Athens, 75, M. Asias Street, 11527, Athens, Greece

      Eva Kassi, Narjes Nasiri-Ansari, Eliana Spilioti, Paraskevi Moutsatsou & Athanasios G. Papavassiliou

    2. First Department of Internal Medicine, Medical School, National and Kapodistrian University of Athens, ‘Laikon’ General Hospital, 11527, Athens, Greece

      Vassiliki Kalotychou

    3. Center for Basic Research, Biomedical Research Foundation of the Academy of Athens, 11527, Athens, Greece

      Panagiota E. Apostolou

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    1. Eva Kassi
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    E. Kassi and N. Nasiri-Ansari contributed equally to this work.

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    Kassi, E., Nasiri-Ansari, N., Spilioti, E. et al. Vitamin D interferes with glucocorticoid responsiveness in human peripheral blood mononuclear target cells. Cell. Mol. Life Sci. 73, 4341–4354 (2016). https://doi.org/10.1007/s00018-016-2281-3

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