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Vitamin D up-regulates the vitamin D receptor by protecting it from proteasomal degradation in human CD4+ T cells.

Kongsbak M, von Essen MR, Boding L, Levring TB, Schjerling P, Lauritsen JP, Woetmann A, Ødum N, Bonefeld CM, Geisler C - PLoS ONE (2014)

Bottom Line: The active form of vitamin D3, 1,25(OH)2D3, has significant immunomodulatory properties and is an important determinant in the differentiation of CD4+ effector T cells.We found that activated CD4+ T cells have the capacity to convert the inactive 25(OH)D3 to the active 1,25(OH)2D3 that subsequently up-regulates VDR protein expression approximately 2-fold. 1,25(OH)2D3 does not increase VDR mRNA expression but increases the half-life of the VDR protein in activated CD4+ T cells.In conclusion, our study shows that activated CD4+ T cells can produce 1,25(OH)2D3, and that 1,25(OH)2D3 induces a 2-fold up-regulation of the VDR protein expression in activated CD4+ T cells by protecting the VDR against proteasomal degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

ABSTRACT
The active form of vitamin D3, 1,25(OH)2D3, has significant immunomodulatory properties and is an important determinant in the differentiation of CD4+ effector T cells. The biological actions of 1,25(OH)2D3 are mediated by the vitamin D receptor (VDR) and are believed to correlate with the VDR protein expression level in a given cell. The aim of this study was to determine if and how 1,25(OH)2D3 by itself regulates VDR expression in human CD4+ T cells. We found that activated CD4+ T cells have the capacity to convert the inactive 25(OH)D3 to the active 1,25(OH)2D3 that subsequently up-regulates VDR protein expression approximately 2-fold. 1,25(OH)2D3 does not increase VDR mRNA expression but increases the half-life of the VDR protein in activated CD4+ T cells. Furthermore, 1,25(OH)2D3 induces a significant intracellular redistribution of the VDR. We show that 1,25(OH)2D3 stabilizes the VDR by protecting it from proteasomal degradation. Finally, we demonstrate that proteasome inhibition leads to up-regulation of VDR protein expression and increases 1,25(OH)2D3-induced gene activation. In conclusion, our study shows that activated CD4+ T cells can produce 1,25(OH)2D3, and that 1,25(OH)2D3 induces a 2-fold up-regulation of the VDR protein expression in activated CD4+ T cells by protecting the VDR against proteasomal degradation.

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Activated human CD4+ T cells produce 1,25(OH)2D3 and up-regulates VDR expression in the presence of 25(OH)D3.(A) 1,25(OH)2D3 in the supernatants of activated and unstimulated T cells and in cell free cultures incubated with the indicated concentrations of 25(OH)D3. Mean ± SEM (n = 5). (B) Representative Western blot of VDR and CD3ζ (loading control) expression in naïve and activated T cells incubated in the presence of the indicated concentrations (nM) of 25(OH)D3. (C) Relative VDR protein expression as determined by the density of the VDR bands from Western blots of naïve and activated T cells incubated in the presence of the indicated concentrations (nM) of 25(OH)D3. The density of the VDR bands were normalized to the density of the VDR bands of T cells activated in the absence of 25(OH)D3. Mean + SEM (n = 7; * p<0.05). (D) Relative VDR mRNA expression in naïve and activated T cells incubated in the presence of the indicated concentrations (nM) of 25(OH)D3. The VDR mRNA levels were normalized to VDR mRNA levels of T cells activated in the absence of 25(OH)D3. Mean + SEM (n = 4; * p<0.001). (E) Relative CYP24A1 mRNA expression in naïve and activated T cells incubated in the presence of the indicated concentrations (nM) of 25(OH)D3. The CYP24A1 mRNA levels were normalized to CYP24A1 mRNA levels of T cells activated in the absence of 25(OH)D3. Mean + SEM (n = 3; * p<0.05). (F) Representative Western blot of VDR and CD3ζ (loading control) expression in T cells activated for 3 days in the presence of polarizing cytokines and anti-cytokine antibodies as indicated. (G) Relative VDR protein expression as determined by the density of the VDR bands from Western blots of T cells treated as described in F. The density of the VDR bands were normalized to the density of the VDR bands of T cells activated in the absence of polarizing cytokines and anti-cytokine antibodies (Th0). Mean + SEM (n = 2).
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pone-0096695-g001: Activated human CD4+ T cells produce 1,25(OH)2D3 and up-regulates VDR expression in the presence of 25(OH)D3.(A) 1,25(OH)2D3 in the supernatants of activated and unstimulated T cells and in cell free cultures incubated with the indicated concentrations of 25(OH)D3. Mean ± SEM (n = 5). (B) Representative Western blot of VDR and CD3ζ (loading control) expression in naïve and activated T cells incubated in the presence of the indicated concentrations (nM) of 25(OH)D3. (C) Relative VDR protein expression as determined by the density of the VDR bands from Western blots of naïve and activated T cells incubated in the presence of the indicated concentrations (nM) of 25(OH)D3. The density of the VDR bands were normalized to the density of the VDR bands of T cells activated in the absence of 25(OH)D3. Mean + SEM (n = 7; * p<0.05). (D) Relative VDR mRNA expression in naïve and activated T cells incubated in the presence of the indicated concentrations (nM) of 25(OH)D3. The VDR mRNA levels were normalized to VDR mRNA levels of T cells activated in the absence of 25(OH)D3. Mean + SEM (n = 4; * p<0.001). (E) Relative CYP24A1 mRNA expression in naïve and activated T cells incubated in the presence of the indicated concentrations (nM) of 25(OH)D3. The CYP24A1 mRNA levels were normalized to CYP24A1 mRNA levels of T cells activated in the absence of 25(OH)D3. Mean + SEM (n = 3; * p<0.05). (F) Representative Western blot of VDR and CD3ζ (loading control) expression in T cells activated for 3 days in the presence of polarizing cytokines and anti-cytokine antibodies as indicated. (G) Relative VDR protein expression as determined by the density of the VDR bands from Western blots of T cells treated as described in F. The density of the VDR bands were normalized to the density of the VDR bands of T cells activated in the absence of polarizing cytokines and anti-cytokine antibodies (Th0). Mean + SEM (n = 2).

Mentions: 25-hydroxyvitamin D3 (25(OH)D3) is the inactive precursor of the active form of vitamin D3, 1,25(OH)2D3, and is considered the most reliant parameter when determining the vitamin D status of a subject. The normal range for serum concentrations of 25(OH)D3 is 25–170 nM, whereas the range for serum concentrations of 1,25(OH)2D3 is 60–110 pM, approximately 1000-fold lower than 25(OH)D3[61]. It has been reported that T cells, especially following activation, express the 25(OH)D3 1α-hydroxylase CYP27B1 that converts the inactive 25(OH)D3 to the active 1,25(OH)2D3; however, whether T cells can convert 25(OH)D3 to 1,25(OH)2D3 in physiological relevant concentrations is a matter of debate [55], [62]. To study whether 25(OH)D3 in physiological concentrations affects VDR expression, we first analyzed whether T cells actually had the ability to produce 1,25(OH)2D3 from 25(OH)D3 in our experimental setup. We purified naïve CD4+ T cells and either left them unstimulated or stimulated them with CD3/CD28 beads in the presence of increasing concentrations of 25(OH)D3. After 3 days we measured the concentration of 1,25(OH)2D3 in the supernatants. Activated T cells clearly had the ability to convert 25(OH)D3 to 1,25(OH)2D3 and produced significant amounts of 1,25(OH)2D3 compared to unstimulated T cells (Fig. 1A). In cell free control samples with 25(OH)D3 but without T cells, 1,25(OH)2D3 could not be detected (Fig. 1A). These results demonstrate that activated human CD4+ T cells have the capacity to produce 1,25(OH)2D3 from 25(OH)D3. To study how 1,25(OH)2D3 affects VDR expression levels, we determined VDR protein expression by Western blot analysis of T cells activated in the presence of increasing concentrations of 25(OH)D3. We found that T cell activation clearly induced VDR protein expression even in the absence of added 25(OH)D3 (Fig. 1B). Interestingly, 25(OH)D3 significantly increased the expression of the VDR in parallel with the 1,25(OH)2D3 production (Fig. 1A–C). Compared to T cells activated in the absence of 25(OH)D3, VDR protein expression was increased 2.0–2.3 fold in T cells activated in the presence of physiological concentrations of 25(OH)D3 at 33 – 100 nM (Fig. 1C). Naive T cells did not express the VDR, not even in the presence of 25(OH)D3 (Fig. 1B and data not shown).


Vitamin D up-regulates the vitamin D receptor by protecting it from proteasomal degradation in human CD4+ T cells.

Kongsbak M, von Essen MR, Boding L, Levring TB, Schjerling P, Lauritsen JP, Woetmann A, Ødum N, Bonefeld CM, Geisler C - PLoS ONE (2014)

Activated human CD4+ T cells produce 1,25(OH)2D3 and up-regulates VDR expression in the presence of 25(OH)D3.(A) 1,25(OH)2D3 in the supernatants of activated and unstimulated T cells and in cell free cultures incubated with the indicated concentrations of 25(OH)D3. Mean ± SEM (n = 5). (B) Representative Western blot of VDR and CD3ζ (loading control) expression in naïve and activated T cells incubated in the presence of the indicated concentrations (nM) of 25(OH)D3. (C) Relative VDR protein expression as determined by the density of the VDR bands from Western blots of naïve and activated T cells incubated in the presence of the indicated concentrations (nM) of 25(OH)D3. The density of the VDR bands were normalized to the density of the VDR bands of T cells activated in the absence of 25(OH)D3. Mean + SEM (n = 7; * p<0.05). (D) Relative VDR mRNA expression in naïve and activated T cells incubated in the presence of the indicated concentrations (nM) of 25(OH)D3. The VDR mRNA levels were normalized to VDR mRNA levels of T cells activated in the absence of 25(OH)D3. Mean + SEM (n = 4; * p<0.001). (E) Relative CYP24A1 mRNA expression in naïve and activated T cells incubated in the presence of the indicated concentrations (nM) of 25(OH)D3. The CYP24A1 mRNA levels were normalized to CYP24A1 mRNA levels of T cells activated in the absence of 25(OH)D3. Mean + SEM (n = 3; * p<0.05). (F) Representative Western blot of VDR and CD3ζ (loading control) expression in T cells activated for 3 days in the presence of polarizing cytokines and anti-cytokine antibodies as indicated. (G) Relative VDR protein expression as determined by the density of the VDR bands from Western blots of T cells treated as described in F. The density of the VDR bands were normalized to the density of the VDR bands of T cells activated in the absence of polarizing cytokines and anti-cytokine antibodies (Th0). Mean + SEM (n = 2).
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pone-0096695-g001: Activated human CD4+ T cells produce 1,25(OH)2D3 and up-regulates VDR expression in the presence of 25(OH)D3.(A) 1,25(OH)2D3 in the supernatants of activated and unstimulated T cells and in cell free cultures incubated with the indicated concentrations of 25(OH)D3. Mean ± SEM (n = 5). (B) Representative Western blot of VDR and CD3ζ (loading control) expression in naïve and activated T cells incubated in the presence of the indicated concentrations (nM) of 25(OH)D3. (C) Relative VDR protein expression as determined by the density of the VDR bands from Western blots of naïve and activated T cells incubated in the presence of the indicated concentrations (nM) of 25(OH)D3. The density of the VDR bands were normalized to the density of the VDR bands of T cells activated in the absence of 25(OH)D3. Mean + SEM (n = 7; * p<0.05). (D) Relative VDR mRNA expression in naïve and activated T cells incubated in the presence of the indicated concentrations (nM) of 25(OH)D3. The VDR mRNA levels were normalized to VDR mRNA levels of T cells activated in the absence of 25(OH)D3. Mean + SEM (n = 4; * p<0.001). (E) Relative CYP24A1 mRNA expression in naïve and activated T cells incubated in the presence of the indicated concentrations (nM) of 25(OH)D3. The CYP24A1 mRNA levels were normalized to CYP24A1 mRNA levels of T cells activated in the absence of 25(OH)D3. Mean + SEM (n = 3; * p<0.05). (F) Representative Western blot of VDR and CD3ζ (loading control) expression in T cells activated for 3 days in the presence of polarizing cytokines and anti-cytokine antibodies as indicated. (G) Relative VDR protein expression as determined by the density of the VDR bands from Western blots of T cells treated as described in F. The density of the VDR bands were normalized to the density of the VDR bands of T cells activated in the absence of polarizing cytokines and anti-cytokine antibodies (Th0). Mean + SEM (n = 2).
Mentions: 25-hydroxyvitamin D3 (25(OH)D3) is the inactive precursor of the active form of vitamin D3, 1,25(OH)2D3, and is considered the most reliant parameter when determining the vitamin D status of a subject. The normal range for serum concentrations of 25(OH)D3 is 25–170 nM, whereas the range for serum concentrations of 1,25(OH)2D3 is 60–110 pM, approximately 1000-fold lower than 25(OH)D3[61]. It has been reported that T cells, especially following activation, express the 25(OH)D3 1α-hydroxylase CYP27B1 that converts the inactive 25(OH)D3 to the active 1,25(OH)2D3; however, whether T cells can convert 25(OH)D3 to 1,25(OH)2D3 in physiological relevant concentrations is a matter of debate [55], [62]. To study whether 25(OH)D3 in physiological concentrations affects VDR expression, we first analyzed whether T cells actually had the ability to produce 1,25(OH)2D3 from 25(OH)D3 in our experimental setup. We purified naïve CD4+ T cells and either left them unstimulated or stimulated them with CD3/CD28 beads in the presence of increasing concentrations of 25(OH)D3. After 3 days we measured the concentration of 1,25(OH)2D3 in the supernatants. Activated T cells clearly had the ability to convert 25(OH)D3 to 1,25(OH)2D3 and produced significant amounts of 1,25(OH)2D3 compared to unstimulated T cells (Fig. 1A). In cell free control samples with 25(OH)D3 but without T cells, 1,25(OH)2D3 could not be detected (Fig. 1A). These results demonstrate that activated human CD4+ T cells have the capacity to produce 1,25(OH)2D3 from 25(OH)D3. To study how 1,25(OH)2D3 affects VDR expression levels, we determined VDR protein expression by Western blot analysis of T cells activated in the presence of increasing concentrations of 25(OH)D3. We found that T cell activation clearly induced VDR protein expression even in the absence of added 25(OH)D3 (Fig. 1B). Interestingly, 25(OH)D3 significantly increased the expression of the VDR in parallel with the 1,25(OH)2D3 production (Fig. 1A–C). Compared to T cells activated in the absence of 25(OH)D3, VDR protein expression was increased 2.0–2.3 fold in T cells activated in the presence of physiological concentrations of 25(OH)D3 at 33 – 100 nM (Fig. 1C). Naive T cells did not express the VDR, not even in the presence of 25(OH)D3 (Fig. 1B and data not shown).

Bottom Line: The active form of vitamin D3, 1,25(OH)2D3, has significant immunomodulatory properties and is an important determinant in the differentiation of CD4+ effector T cells.We found that activated CD4+ T cells have the capacity to convert the inactive 25(OH)D3 to the active 1,25(OH)2D3 that subsequently up-regulates VDR protein expression approximately 2-fold. 1,25(OH)2D3 does not increase VDR mRNA expression but increases the half-life of the VDR protein in activated CD4+ T cells.In conclusion, our study shows that activated CD4+ T cells can produce 1,25(OH)2D3, and that 1,25(OH)2D3 induces a 2-fold up-regulation of the VDR protein expression in activated CD4+ T cells by protecting the VDR against proteasomal degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

ABSTRACT
The active form of vitamin D3, 1,25(OH)2D3, has significant immunomodulatory properties and is an important determinant in the differentiation of CD4+ effector T cells. The biological actions of 1,25(OH)2D3 are mediated by the vitamin D receptor (VDR) and are believed to correlate with the VDR protein expression level in a given cell. The aim of this study was to determine if and how 1,25(OH)2D3 by itself regulates VDR expression in human CD4+ T cells. We found that activated CD4+ T cells have the capacity to convert the inactive 25(OH)D3 to the active 1,25(OH)2D3 that subsequently up-regulates VDR protein expression approximately 2-fold. 1,25(OH)2D3 does not increase VDR mRNA expression but increases the half-life of the VDR protein in activated CD4+ T cells. Furthermore, 1,25(OH)2D3 induces a significant intracellular redistribution of the VDR. We show that 1,25(OH)2D3 stabilizes the VDR by protecting it from proteasomal degradation. Finally, we demonstrate that proteasome inhibition leads to up-regulation of VDR protein expression and increases 1,25(OH)2D3-induced gene activation. In conclusion, our study shows that activated CD4+ T cells can produce 1,25(OH)2D3, and that 1,25(OH)2D3 induces a 2-fold up-regulation of the VDR protein expression in activated CD4+ T cells by protecting the VDR against proteasomal degradation.

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Related in: MedlinePlus