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1alpha,25-dihydroxyvitamin D(3)-induced myeloid cell differentiation is regulated by a vitamin D receptor-phosphatidylinositol 3-kinase signaling complex.

Hmama Z, Nandan D, Sly L, Knutson KL, Herrera-Velit P, Reiner NE - J. Exp. Med. (1999)

Bottom Line: This was associated with rapid and transient increases in phosphatidylinositol 3-kinase (PI 3-kinase) activity.In THP-1 cells, induction of CD11b expression by D(3) was also abrogated by LY294002 and wortmannin.In contrast to CD14 and CD11b, hormone-induced expression of the Cdk inhibitor p21 in THP-1 cells was unaffected by either wortmannin or LY294002.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, The University of British Columbia, Faculties of Medicine and Science, The Research Institute of the Vancouver Hospital and Health Sciences Center, Vancouver, British Columbia, Canada.

ABSTRACT
1alpha,25-dihydroxyvitamin D(3) (D(3)) promotes the maturation of myeloid cells and surface expressions of CD14 and CD11b, markers of cell differentiation in response to D(3). To examine how these responses are regulated, THP-1 cells were grown in serum-free medium and incubated with D(3). This was associated with rapid and transient increases in phosphatidylinositol 3-kinase (PI 3-kinase) activity. Furthermore, induction of CD14 expression in response to D(3) was abrogated by (a) the PI 3-kinase inhibitors LY294002 and wortmannin; (b) antisense oligonucleotides to mRNA for the p110 catalytic subunit of PI 3-kinase; and (c) a dominant negative mutant of PI 3-kinase. In THP-1 cells, induction of CD11b expression by D(3) was also abrogated by LY294002 and wortmannin. Similarly, LY294002 and wortmannin inhibited D(3)-induced expression of both CD14 and CD11b in peripheral blood monocytes. In contrast to CD14 and CD11b, hormone-induced expression of the Cdk inhibitor p21 in THP-1 cells was unaffected by either wortmannin or LY294002. These findings suggest that PI 3-kinase selectively regulates D(3)-induced monocyte differentiation, independent of any effects on p21.

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Wortmannin and LY294002 attenuate D3-induced CD14 expression, but not expression of Cdk inhibitor p21. Serum-starved THP-1 (4 × 106) cells were incubated for 20 min at 37°C and 5% CO2 in medium alone, with wortmannin (Wrtm), or with LY294002 (LY). D3 (100 nM) was then added for 24 h at 37°C. (A) Aliquots of cells (∼0.5 × 106) from each treatment were washed with staining buffer and labeled with anti-CD14 mAb or irrelevant mAb, followed by FITC-conjugated secondary Abs. Flow cytometric analyses were performed, and MFI indices were determined as described in the legend to Fig. 1. The results shown are the means ± SEM of values obtained in three separate experiments. (B and C) Total RNA was extracted from the cells remaining, and RT-PCR was carried out for CD14 (B) and p21 (C) as described (reference 41). β-actin was analyzed to control for loading. Negative controls consisting of no RNA and RNA without RT were included, and these produced no signals (data not shown). The data shown are from one of two independent experiments that yielded similar results.
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Figure 3: Wortmannin and LY294002 attenuate D3-induced CD14 expression, but not expression of Cdk inhibitor p21. Serum-starved THP-1 (4 × 106) cells were incubated for 20 min at 37°C and 5% CO2 in medium alone, with wortmannin (Wrtm), or with LY294002 (LY). D3 (100 nM) was then added for 24 h at 37°C. (A) Aliquots of cells (∼0.5 × 106) from each treatment were washed with staining buffer and labeled with anti-CD14 mAb or irrelevant mAb, followed by FITC-conjugated secondary Abs. Flow cytometric analyses were performed, and MFI indices were determined as described in the legend to Fig. 1. The results shown are the means ± SEM of values obtained in three separate experiments. (B and C) Total RNA was extracted from the cells remaining, and RT-PCR was carried out for CD14 (B) and p21 (C) as described (reference 41). β-actin was analyzed to control for loading. Negative controls consisting of no RNA and RNA without RT were included, and these produced no signals (data not shown). The data shown are from one of two independent experiments that yielded similar results.

Mentions: To address whether PI 3-kinase activation is required for D3-induced monocyte differentiation, serum-starved THP-1 cells were incubated with either wortmannin or LY294002 for 20 min before addition of hormone. Inhibitors were used at concentrations known to be relatively selective for inhibition of PI 3-kinase 4850. Incubation with inhibitors alone had no effect on basal expression of CD14 (data not shown). Preincubation with 5 nM wortmannin led to 65.7 ± 5.6% inhibition of D3-induced CD14 expression (mean ± SEM, n = 3; Fig. 3 A). Increasing the concentration of wortmannin to 50 nM inhibited CD14 expression by 86.0 ± 8.5%. LY294002, an inhibitor of PI 3-kinase that acts via a distinct mechanism from that of wortmannin, when used at 1.5 μM reduced D3-induced CD14 expression by 66.8 ± 11.9%. CD14 expression decreased further in the presence of 15 μM LY294002 (88.1 ± 5.1%). In contrast to abrogation of D3-induced CD14, neither wortmannin nor LY294002 had significant effects on the expression of HLA class I molecules (data not shown), indicating that the effects of neither wortmannin nor LY294002 were due to nonspecific toxicity. The PI 3-kinase inhibitors LY294002 and wortmannin also attenuated D3-induced cell surface expression of CD11b by THP-1 cells. While incubation with inhibitors alone had no effect on basal levels of CD11b expression, preincubation with 5 nM wortmannin inhibited the response to D3 by 41 ± 7% (Table ). A higher concentration of 50 nM increased inhibition to 100 ± 12%. LY294002 inhibited D3-induced CD11b surface expression by 48 ± 10% at a concentration of 1.5 μM, and by 88 ± 14% at a concentration of 15 μM (Table ). Surface expression of CD14 and CD11b was also examined in human peripheral blood monocytes. As shown in Table , LY294002 and wortmannin markedly inhibited D3-induced expression of both CD14 and CD11b in these cells.


1alpha,25-dihydroxyvitamin D(3)-induced myeloid cell differentiation is regulated by a vitamin D receptor-phosphatidylinositol 3-kinase signaling complex.

Hmama Z, Nandan D, Sly L, Knutson KL, Herrera-Velit P, Reiner NE - J. Exp. Med. (1999)

Wortmannin and LY294002 attenuate D3-induced CD14 expression, but not expression of Cdk inhibitor p21. Serum-starved THP-1 (4 × 106) cells were incubated for 20 min at 37°C and 5% CO2 in medium alone, with wortmannin (Wrtm), or with LY294002 (LY). D3 (100 nM) was then added for 24 h at 37°C. (A) Aliquots of cells (∼0.5 × 106) from each treatment were washed with staining buffer and labeled with anti-CD14 mAb or irrelevant mAb, followed by FITC-conjugated secondary Abs. Flow cytometric analyses were performed, and MFI indices were determined as described in the legend to Fig. 1. The results shown are the means ± SEM of values obtained in three separate experiments. (B and C) Total RNA was extracted from the cells remaining, and RT-PCR was carried out for CD14 (B) and p21 (C) as described (reference 41). β-actin was analyzed to control for loading. Negative controls consisting of no RNA and RNA without RT were included, and these produced no signals (data not shown). The data shown are from one of two independent experiments that yielded similar results.
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Figure 3: Wortmannin and LY294002 attenuate D3-induced CD14 expression, but not expression of Cdk inhibitor p21. Serum-starved THP-1 (4 × 106) cells were incubated for 20 min at 37°C and 5% CO2 in medium alone, with wortmannin (Wrtm), or with LY294002 (LY). D3 (100 nM) was then added for 24 h at 37°C. (A) Aliquots of cells (∼0.5 × 106) from each treatment were washed with staining buffer and labeled with anti-CD14 mAb or irrelevant mAb, followed by FITC-conjugated secondary Abs. Flow cytometric analyses were performed, and MFI indices were determined as described in the legend to Fig. 1. The results shown are the means ± SEM of values obtained in three separate experiments. (B and C) Total RNA was extracted from the cells remaining, and RT-PCR was carried out for CD14 (B) and p21 (C) as described (reference 41). β-actin was analyzed to control for loading. Negative controls consisting of no RNA and RNA without RT were included, and these produced no signals (data not shown). The data shown are from one of two independent experiments that yielded similar results.
Mentions: To address whether PI 3-kinase activation is required for D3-induced monocyte differentiation, serum-starved THP-1 cells were incubated with either wortmannin or LY294002 for 20 min before addition of hormone. Inhibitors were used at concentrations known to be relatively selective for inhibition of PI 3-kinase 4850. Incubation with inhibitors alone had no effect on basal expression of CD14 (data not shown). Preincubation with 5 nM wortmannin led to 65.7 ± 5.6% inhibition of D3-induced CD14 expression (mean ± SEM, n = 3; Fig. 3 A). Increasing the concentration of wortmannin to 50 nM inhibited CD14 expression by 86.0 ± 8.5%. LY294002, an inhibitor of PI 3-kinase that acts via a distinct mechanism from that of wortmannin, when used at 1.5 μM reduced D3-induced CD14 expression by 66.8 ± 11.9%. CD14 expression decreased further in the presence of 15 μM LY294002 (88.1 ± 5.1%). In contrast to abrogation of D3-induced CD14, neither wortmannin nor LY294002 had significant effects on the expression of HLA class I molecules (data not shown), indicating that the effects of neither wortmannin nor LY294002 were due to nonspecific toxicity. The PI 3-kinase inhibitors LY294002 and wortmannin also attenuated D3-induced cell surface expression of CD11b by THP-1 cells. While incubation with inhibitors alone had no effect on basal levels of CD11b expression, preincubation with 5 nM wortmannin inhibited the response to D3 by 41 ± 7% (Table ). A higher concentration of 50 nM increased inhibition to 100 ± 12%. LY294002 inhibited D3-induced CD11b surface expression by 48 ± 10% at a concentration of 1.5 μM, and by 88 ± 14% at a concentration of 15 μM (Table ). Surface expression of CD14 and CD11b was also examined in human peripheral blood monocytes. As shown in Table , LY294002 and wortmannin markedly inhibited D3-induced expression of both CD14 and CD11b in these cells.

Bottom Line: This was associated with rapid and transient increases in phosphatidylinositol 3-kinase (PI 3-kinase) activity.In THP-1 cells, induction of CD11b expression by D(3) was also abrogated by LY294002 and wortmannin.In contrast to CD14 and CD11b, hormone-induced expression of the Cdk inhibitor p21 in THP-1 cells was unaffected by either wortmannin or LY294002.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, The University of British Columbia, Faculties of Medicine and Science, The Research Institute of the Vancouver Hospital and Health Sciences Center, Vancouver, British Columbia, Canada.

ABSTRACT
1alpha,25-dihydroxyvitamin D(3) (D(3)) promotes the maturation of myeloid cells and surface expressions of CD14 and CD11b, markers of cell differentiation in response to D(3). To examine how these responses are regulated, THP-1 cells were grown in serum-free medium and incubated with D(3). This was associated with rapid and transient increases in phosphatidylinositol 3-kinase (PI 3-kinase) activity. Furthermore, induction of CD14 expression in response to D(3) was abrogated by (a) the PI 3-kinase inhibitors LY294002 and wortmannin; (b) antisense oligonucleotides to mRNA for the p110 catalytic subunit of PI 3-kinase; and (c) a dominant negative mutant of PI 3-kinase. In THP-1 cells, induction of CD11b expression by D(3) was also abrogated by LY294002 and wortmannin. Similarly, LY294002 and wortmannin inhibited D(3)-induced expression of both CD14 and CD11b in peripheral blood monocytes. In contrast to CD14 and CD11b, hormone-induced expression of the Cdk inhibitor p21 in THP-1 cells was unaffected by either wortmannin or LY294002. These findings suggest that PI 3-kinase selectively regulates D(3)-induced monocyte differentiation, independent of any effects on p21.

Show MeSH
Related in: MedlinePlus