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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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VDR antisense S-oligo inhibits D3-induced CD14 expression. 5 × 106 THP-1 cells were suspended in 500 μl of 2.5% lipofectAMINE/RPMI 1640 alone (control), or containing 5 μM of either sense (S) or antisense (AS) S-oligo to the VDR. Cells were then incubated on a rotary shaker for 4 h at 37°C. The volume was then brought up to 7 ml, and cells were cultured for an additional 18 h at 37°C and 5% CO2. (A) 0.5 × 106 cells were boiled in Laemmli buffer and subjected to SDS-PAGE and immunoblotting with mAb to VDR. Membranes were developed by ECL as described (reference 44). The data shown are from one of two independent experiments that yielded similar results. (B) Aliquots of control or S-oligo–treated cells (∼1 × 106) were incubated with D3 (100 nM, 24 h), washed with staining buffer, and then 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. Results are expressed as histograms of fluorescence intensity. Histograms displaced to the right represent cells stained with anti-CD14, and histograms on the left represent cells stained with irrelevant IgG2b. Bold, italicized values in each frame are averages (n = 2) of MFI indices, determined as in the legend to Fig. 1. (C) Control or S-oligo–treated cells (∼3 × 106) were incubated with D3 (100 nM, 24 h). Total RNA was extracted and RT-PCR was carried out for CD14 and β-actin as described (reference 41). Controls consisting of no RNA and RNA without RT were included, and no signals were obtained (data not shown). The data shown are from one of two independent experiments that yielded similar results.
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Figure 6: VDR antisense S-oligo inhibits D3-induced CD14 expression. 5 × 106 THP-1 cells were suspended in 500 μl of 2.5% lipofectAMINE/RPMI 1640 alone (control), or containing 5 μM of either sense (S) or antisense (AS) S-oligo to the VDR. Cells were then incubated on a rotary shaker for 4 h at 37°C. The volume was then brought up to 7 ml, and cells were cultured for an additional 18 h at 37°C and 5% CO2. (A) 0.5 × 106 cells were boiled in Laemmli buffer and subjected to SDS-PAGE and immunoblotting with mAb to VDR. Membranes were developed by ECL as described (reference 44). The data shown are from one of two independent experiments that yielded similar results. (B) Aliquots of control or S-oligo–treated cells (∼1 × 106) were incubated with D3 (100 nM, 24 h), washed with staining buffer, and then 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. Results are expressed as histograms of fluorescence intensity. Histograms displaced to the right represent cells stained with anti-CD14, and histograms on the left represent cells stained with irrelevant IgG2b. Bold, italicized values in each frame are averages (n = 2) of MFI indices, determined as in the legend to Fig. 1. (C) Control or S-oligo–treated cells (∼3 × 106) were incubated with D3 (100 nM, 24 h). Total RNA was extracted and RT-PCR was carried out for CD14 and β-actin as described (reference 41). Controls consisting of no RNA and RNA without RT were included, and no signals were obtained (data not shown). The data shown are from one of two independent experiments that yielded similar results.

Mentions: Many responses to D3, such as induction of expression of osteopontin, osteocalcin, calbindin, and 24-hydroxylase, are brought about by a mechanism involving binding of the VDR to a specific VDRE in the corresponding promoters 53. Since no VDRE has been identified within the CD14 gene promoter 28, this raised the question as to whether the VDR plays any role in regulating D3-induced CD14 expression. To address this question, THP-1 cells were incubated overnight with antisense S-oligo specific to VDR mRNA. This resulted in significant attenuation of the level of VDR protein in THP-1 cells as detected by Western blotting (Fig. 6 A). In contrast, pretreatment of cells with the control, sense S-oligo had no apparent effect on the level of VDR protein. In parallel with the reduction of VDR protein, D3-induced surface expression of CD14 was markedly attenuated (Fig. 6 B; in two separate experiments an average decrease of 86.8% was observed). In addition, cells were treated with S-oligo antisense specific for VDR mRNA before D3, followed by RNA extraction and RT-PCR. The results showed that the induction of CD14 mRNA was markedly attenuated in antisense-treated cells (Fig. 6 C). Taken together, these findings strongly suggest that the VDR is essential for D3-induced CD14 gene expression.


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)

VDR antisense S-oligo inhibits D3-induced CD14 expression. 5 × 106 THP-1 cells were suspended in 500 μl of 2.5% lipofectAMINE/RPMI 1640 alone (control), or containing 5 μM of either sense (S) or antisense (AS) S-oligo to the VDR. Cells were then incubated on a rotary shaker for 4 h at 37°C. The volume was then brought up to 7 ml, and cells were cultured for an additional 18 h at 37°C and 5% CO2. (A) 0.5 × 106 cells were boiled in Laemmli buffer and subjected to SDS-PAGE and immunoblotting with mAb to VDR. Membranes were developed by ECL as described (reference 44). The data shown are from one of two independent experiments that yielded similar results. (B) Aliquots of control or S-oligo–treated cells (∼1 × 106) were incubated with D3 (100 nM, 24 h), washed with staining buffer, and then 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. Results are expressed as histograms of fluorescence intensity. Histograms displaced to the right represent cells stained with anti-CD14, and histograms on the left represent cells stained with irrelevant IgG2b. Bold, italicized values in each frame are averages (n = 2) of MFI indices, determined as in the legend to Fig. 1. (C) Control or S-oligo–treated cells (∼3 × 106) were incubated with D3 (100 nM, 24 h). Total RNA was extracted and RT-PCR was carried out for CD14 and β-actin as described (reference 41). Controls consisting of no RNA and RNA without RT were included, and no signals were obtained (data not shown). The data shown are from one of two independent experiments that yielded similar results.
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Figure 6: VDR antisense S-oligo inhibits D3-induced CD14 expression. 5 × 106 THP-1 cells were suspended in 500 μl of 2.5% lipofectAMINE/RPMI 1640 alone (control), or containing 5 μM of either sense (S) or antisense (AS) S-oligo to the VDR. Cells were then incubated on a rotary shaker for 4 h at 37°C. The volume was then brought up to 7 ml, and cells were cultured for an additional 18 h at 37°C and 5% CO2. (A) 0.5 × 106 cells were boiled in Laemmli buffer and subjected to SDS-PAGE and immunoblotting with mAb to VDR. Membranes were developed by ECL as described (reference 44). The data shown are from one of two independent experiments that yielded similar results. (B) Aliquots of control or S-oligo–treated cells (∼1 × 106) were incubated with D3 (100 nM, 24 h), washed with staining buffer, and then 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. Results are expressed as histograms of fluorescence intensity. Histograms displaced to the right represent cells stained with anti-CD14, and histograms on the left represent cells stained with irrelevant IgG2b. Bold, italicized values in each frame are averages (n = 2) of MFI indices, determined as in the legend to Fig. 1. (C) Control or S-oligo–treated cells (∼3 × 106) were incubated with D3 (100 nM, 24 h). Total RNA was extracted and RT-PCR was carried out for CD14 and β-actin as described (reference 41). Controls consisting of no RNA and RNA without RT were included, and no signals were obtained (data not shown). The data shown are from one of two independent experiments that yielded similar results.
Mentions: Many responses to D3, such as induction of expression of osteopontin, osteocalcin, calbindin, and 24-hydroxylase, are brought about by a mechanism involving binding of the VDR to a specific VDRE in the corresponding promoters 53. Since no VDRE has been identified within the CD14 gene promoter 28, this raised the question as to whether the VDR plays any role in regulating D3-induced CD14 expression. To address this question, THP-1 cells were incubated overnight with antisense S-oligo specific to VDR mRNA. This resulted in significant attenuation of the level of VDR protein in THP-1 cells as detected by Western blotting (Fig. 6 A). In contrast, pretreatment of cells with the control, sense S-oligo had no apparent effect on the level of VDR protein. In parallel with the reduction of VDR protein, D3-induced surface expression of CD14 was markedly attenuated (Fig. 6 B; in two separate experiments an average decrease of 86.8% was observed). In addition, cells were treated with S-oligo antisense specific for VDR mRNA before D3, followed by RNA extraction and RT-PCR. The results showed that the induction of CD14 mRNA was markedly attenuated in antisense-treated cells (Fig. 6 C). Taken together, these findings strongly suggest that the VDR is essential for D3-induced CD14 gene expression.

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