1α,25-Dihydroxyvitamin D₃–Induced Myeloid Cell Differentiation Is Regulated by a Vitamin D Receptor–Phosphatidylinositol 3-Kinase Signaling Complex

RPMI 1640, HBSS, and penicillin/streptomycin were from StemCell Technologies, Inc. Wortmannin, l-α-phosphatidylinositol, PMSF, leupeptin, pepstatin A, and aprotinin were purchased from Sigma Chemical Co. LY294002 and microcystin were from Calbiochem Corp. Protein A–agarose and electrophoresis reagents were purchased from Bio-Rad Laboratories. [γ-³²P]ATP was from Nycomed Amersham Plc.

The following mAbs were used: 3C10 (mouse IgG2b, anti-CD14 mAb; a gift from Dr. W.C. Van Voorhis, University of Washington, Seattle, WA); W6/32 (mouse IgG_2a, anti–HLA class I mAb; American Type Culture Collection); UB93-3 (mouse mAb to PI 3-kinase; Upstate Biotechnology Inc.) and 9A7 (rat IgG_2b, anti-VDR; Chemicon International). Other Abs used included anti-CD14 and anti-CD11b, both murine IgG₁ (SC-7328 and SC-1186, respectively; Santa Cruz Biotechnology, Inc.). Murine IgG₁ isotype control MG100 was from Caltag Laboratories.

The promonocytic cell line THP-1 was from the American Type Culture Collection. The promonocytic cell line U937 transfected with cDNA, encoding the entire coding region of either wild-type bovine PI 3-kinase subunit p85α (Wp85α) or mutant bovine p85α, (Δp85α) has been described 37. The mutant has a deletion of 35 amino acids from residues 479–513 of bovine p85α and the insertion of two other amino acids (Ser-Arg) in the deleted position. Mutant p85α competes with native p85 for binding to essential signaling proteins, thereby acting as a dominant negative mutant 38. THP-1 and U937 cell lines were cultured in RPMI 1640 supplemented with 10% FCS (HyClone), 2 mM l-glutamine, penicillin (100 U/ml), and streptomycin (100 μg/ml). Cell density was maintained at a concentration of <5 × 10⁵/ml.

Peripheral blood mononuclear cells were isolated as described previously 39. Monocytes were allowed to adhere for 1 h at 37°C in a humidified atmosphere with 5% CO₂. Nonadherent cells were removed by three washes with HBSS. Adherent cells were immediately treated and used for cell surface phenotype analysis.

To measure the expression of surface molecules, cells were incubated with specific mouse mAb or irrelevant isotype-matched IgG (10 μg/ml) for 30 min, then washed twice and labeled with FITC-conjugated F(ab′)₂ sheep anti–mouse IgG (Sigma Chemical Co.) for 30 min. Cells were then washed twice and fixed in 2% paraformaldehyde in staining buffer. All staining and washing procedures were performed at 4°C in HBSS containing 0.1% NaN₃ and 1% FCS. Cell fluorescence was analyzed using a Coulter Elite flow cytometer. Relative fluorescence intensities of 5,000–10,000 cells were recorded as single-parameter histograms (log scale, 1,024 channels, 4 log decades), and the mean fluorescence intensity (MFI) was calculated for each histogram. Results are expressed as MFI indices which correspond to MFI of cells + specific Ab/MFI of cells + irrelevant isotype-matched IgG.

Cell lysates for analysis of PI 3-kinase were prepared in 20 mM Tris, pH 8.0, 1% Triton X-100, 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM Na₃VO₄, 5 mM NaF, 100 nM microcystin, 1 mM PMSF, 10 μg/ml leupeptin, 10 μg/ml pepstatin, and 10 μg/ml aprotinin. Aliquots of lysates adjusted for protein concentration (300–500 μg protein) were incubated for 2–4 h at 4°C with UB93-3 mAb (anti–PI 3-kinase), and immune complexes were adsorbed onto protein A–agarose for 30–60 min. The complexes were washed twice with lysis buffer and three times with 10 mM Tris-HCl, pH 7.4. PI 3-kinase activity was measured as described 22,40. In brief, immunoprecipitates were incubated for 10 min at 4°C with 10 μg of sonicated (3 times for 20 s in an ultrasonic cell disrupter; Branson Sonic Power Co.) l-α-phosphatidylinositol in 10 μl of 30 mM Hepes, to which was added 40 μl of kinase assay buffer (30 mM Hepes, 30 mM MgCl₂, 200 μM adenosine, 50 μM ATP, and 10 μCi of [γ-³²P]ATP). Reactions were carried out for 15 min at room temperature, and stopped by the addition of 100 μl of 1 N HCl and 200 μl of chloroform/methanol (1:1, vol/vol). Lipids were separated on oxalate-treated silica TLC plates using a solvent system of chloroform/methanol/water/28% ammonia (45:35:7.5:2.5, vol/vol/vol/vol). Plates were exposed to X-ray film at −70°C. Incorporation of radioactivity into lipids was measured by excising the corresponding portions of the TLC plate, followed by liquid scintillation counting. Alternatively, cell lysates were incubated overnight at 4°C with 9A7 mAb (anti-VDR), and immune complexes were adsorbed onto protein A–agarose, washed in lysis buffer containing 200 mM NaCl, and assayed for PI 3-kinase activity as described above.

RNA isolation, cDNA synthesis, and PCR conditions were as described previously 41. Sequences (5′ to 3′) of oligonucleotide primers used in PCR amplifications were as follows: CD14 sense, CCC AAG CTT GGG CAG AGG TTC GGA AGA CTT ATC G; CD14 antisense, GGG GTA CCC CTT GAC CGT GTC AGC ATA CTG CC 29; Cdk inhibitor p21 sense, TTC TCC TTT TCC TCT CTC C; p21 antisense, TCT ACT CCC CCA TCA TAT ACC; β-actin sense, CAC CCC GTG CTG CTG ACC GAG GCC; β-actin antisense, CCA CAC GGA GTA CTT GCG CTC AGG 41. Controls included in the reverse transcription (RT)-PCR reactions were no RNA and RNA without RT, and different cycle numbers of PCR reactions were performed to ensure linear cDNA amplification.

Phosphorothioate-modified oligonucleotides (S-oligos) to VDR and p110 subunit of PI 3-kinase were synthesized by Life Technologies, Inc. The oligonucleotides were phosphorothioate-modified to limit intracellular degradation, and purified by high-performance liquid chromatography to remove incomplete synthesis products. 21-mer sequences, including the presumed translation initiation site of human cDNA sequences corresponding to the VDR 42 and the α isoform of the p110 subunit of PI 3-kinase 43, were made in both sense and antisense orientations with the following sequences: VDR sense, 5′-ATG GAG GCA ATG GCG GCC AGC-3′; VDR antisense, 5′-GCT GGC CGC CAT TGC CTC CAT-3′; p110 sense, 5′-ATG CCT CCA AGA CCA TCA TCA-3′; and p110 antisense, 5′-TGA TGA TGG TCT TGG AGG CAT-3′. The sequences were selected by screening for uniqueness using Blast 227, and were also tested for lack of secondary structure and pairing by using Primer Software (v. 2.0). 3–7 × 10⁶ THP-1 cells were suspended in 500 μl RPMI containing 2.5% lipofectAMINE (Life Technologies, Inc.) and 5 μM S-oligo, and incubated on a rotary shaker for 4 h at 37°C. In addition, fluorescent-modified antisense and sense and flow cytometry were used to monitor S-oligo incorporation into cells. After this incubation, the medium was brought up to 5–10 ml, and cells were cultured for an additional 18 h at 37°C and 5% CO₂.

To examine a potential association between the VDR and PI 3-kinase, D₃-treated cells were lysed in the buffer used to prepare cell lysates for PI 3-kinase assay, then immunoprecipitated with specific mAb to VDR. Immunoprecipitates were washed with lysis buffer containing 200 mM NaCl to minimize nonspecific protein–protein interactions. Samples were then analyzed by SDS-PAGE and immunoblotting with Abs to the p85 PI 3-kinase subunit as described previously 40. To measure the effects of cell treatments with antisense S-oligos on protein levels of VDR, whole-cell lysates prepared by boiling in Laemmli buffer were subjected to SDS-PAGE and immunoblotting with the mAb 9A7. Membranes were developed by enhanced chemiluminescence (ECL) as described 44.

Initial experiments involving immunofluorescence and flow cytometric analysis defined the experimental conditions for D₃-induced CD14 expression by THP-1 cells. THP-1 cells maintained in complete medium expressed nearly undetectable levels of CD14 on the surface, and treatment with hormone caused a dose- and time-dependent increase in CD14 expression (Fig. 1). A response was obtained in the presence of as little as 0.1 nM D₃ (Fig. 1 A), and at 100 nM, virtually all cells expressed CD14 at an ∼50-fold increase in MFI. The kinetics of induction of CD14 in response to 100 nM D₃ is shown in Fig. 1 B. CD14 expression increased progressively with time and was maximal (55-fold increase in MFI index, average of 2 separate experiments) after 48 h. Since a 24-h exposure to 100 nM D₃ was sufficient to induce a high level of surface CD14 (53-fold increase in MFI index, average of 2 separate experiments) in nearly 100% of cells, subsequent experiments were carried out under these conditions.

Other experiments examined whether the CD14 response of cells to D₃ (100 nM, 24 h) was serum dependent. Results obtained in three independent experiments (Fig. 1 C) using RPMI 1640 alone showed that THP-1 cells were fully responsive for CD14 induction (MFI index = 104.4 ± 5.1, mean ± SEM). Addition of FCS had only a limited additive effect (MFI index = 125.0 ± 14.1). Conversely, addition of insulin growth factor (IGF)-I a serum component known to influence cell differentiation 30, to D₃ minimally inhibited CD14 expression. These observations suggest that in THP-1 cells, D₃ is capable of inducing CD14 in the absence of any serum factors, and that D₃ delivery to cell surface is independent of serum vitamin D binding protein 45,46.

In light of the important roles played by PI 3-kinase in regulating the differentiation of a variety of cell types 47,48,49, the possibility that D₃-induced monocytic differentiation involves PI 3-kinase was investigated. As shown in Fig. 2 A, incubation of serum-starved THP-1 cells with 10 nM D₃ brought about a significant increase in PI 3-kinase activity (3.07 ± 1.17 fold increase, mean ± SEM, n = 3), with a maximum response observed at 1 μM (6.59 ± 1.47 fold increase). Although PI 3-kinase activity was also significantly increased in IGF-I (100 ng/ml)–treated cells (Fig. 2 A), this was not associated with increased CD14 expression (Fig. 1 C). Time course experiments using 100 nM D₃ (Fig. 2 B) showed that PI 3-kinase activation was detectable by as early as 5 min and reached a maximum level (4.94 ± 0.72 fold increase) by 20 min.

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 48,50. 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.

Inhibition of PI 3-kinase has been shown to affect the transport of some cell surface molecules 51, and thus was a possible mechanism to explain the effects of PI 3-kinase inhibitors on D₃-induced CD14 expression. However, when total RNA was isolated and RT-PCR was performed using primers for CD14 and β-actin, the results showed that mRNA levels for CD14 were markedly reduced in cells incubated with either wortmannin or LY294002 (Fig. 3 B). These findings indicate that inhibition of PI 3-kinase results in attenuation of D₃-induced CD14 gene expression at a pretranslational level.

Treatment of immature myeloid cells with D₃ induces the expression of the Cdk inhibitor p21, and the latter has been shown to regulate gene expression and to promote myleoid cell differentiation 52. Experiments were carried out to examine whether the induction of p21 in response to hormone also involved PI 3-kinase. As shown in Fig. 3 C, THP-1 cells expressed low levels of p21 mRNA in the basal state. As expected, p21 expression was significantly induced in response to incubation of cells with D₃. However, in contrast to the findings with CD14 and CD11b, preincubation of cells with either wortmannin or LY294002 had no effect on hormone-induced expression of p21. These findings suggest that PI 3-kinase selectively regulates monocyte differentiation, independent of any effects on p21.

The role of PI 3-kinase in regulating monocyte differentiation was investigated further by inhibiting the synthesis of the p110 catalytic subunit of PI 3-kinase. THP-1 cells were incubated in the presence of antisense S-oligo complementary to the p110 translation initiation region (including the ATG initiation codon), and then assayed for both PI 3-kinase activation and CD14 expression in response to D₃. Flow cytometric analysis of cells exposed to fluorescein-modified antisense S-oligo, under the same conditions used for unmodified S-oligos, revealed that THP-1 cells readily incorporated foreign DNA (data not shown). As shown in Fig. 4 A, antisense S-oligo to p110 mRNA significantly attenuated D₃-induced PI 3-kinase activity (% inhibition = 87.1 ± 5.6, mean ± SEM, n = 3), whereas at the same concentration, the control sense S-oligo had no apparent effect on the PI 3-kinase response. In parallel with this effect on PI 3-kinase activation, pretreatment with antisense S-oligo (and not with the control, sense oligo) almost completely inhibited hormone-induced surface expression of CD14 (% inhibition = 91.7 ± 3.9, mean ± SEM of three experiments; Fig. 4 B). In addition, RT-PCR results shown in Fig. 4 C demonstrated that D₃-induced mRNA levels for CD14 were markedly diminished in cells treated with antisense S-oligo to p110 mRNA.

The PI 3-kinase requirement for D₃-induced CD14 expression was also examined in U937 cells transfected with a dominant negative mutant of p85 (Δp85). Stable transfection of these cells with Δp85 resulted in a significant reduction of stimulated PI 3-kinase activity 22,37. Exposure of Δp85 U937 to D₃ (100 nM, 48 h) led to only a marginal increase in surface expression of CD14 above baseline. In contrast, cells transfected with wild-type p85 showed significant induction of CD14 expression (Fig. 5 A). In addition, as observed with THP-1 cells, RT-PCR experiments showed that attenuation of PI 3-kinase in U937 cells resulted in markedly reduced response to D₃ for induction of CD14 mRNA (Fig. 5 B). Taken together, these findings strongly suggest that PI 3-kinase plays a central role in D₃-induced monocyte differentiation, and indicate that PI 3-kinase activation is required to induce CD14 expression.

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.

Given the evidence that both PI 3-kinase and the VDR were essential for induction of CD14 in response to D₃, the question examined next was whether the VDR is required for activation of PI 3-kinase. THP-1 cells were treated with antisense S-oligos as described above to reduce VDR protein expression before incubation with D₃ (100 nM, 20 min). Cell lysates were then prepared and assayed for PI 3-kinase activity. The results in Fig. 7 A show that pretreatment with antisense to VDR almost completely abrogated (92.9% inhibition, average of two separate experiments) PI 3-kinase activation in response to D₃. In contrast, treatment with control sense S-oligos to VDR had no inhibitory effect.

The evidence that the D₃ receptor was required for both PI 3-kinase activation and CD14 expression in response to D₃ suggested the possibility that this might involve a signaling complex containing both the VDR and PI 3-kinase. To test this hypothesis, THP-1 cells were incubated with D₃, and immunoprecipitates prepared with mAb to the VDR were assayed for PI 3-kinase activity. The results shown in Fig. 7 B indicate that immunoprecipitates of the VDR prepared from cells activated with D₃ contained PI 3-kinase activity. To examine further whether the VDR associates with PI 3-kinase upon D₃ stimulation, aliquots from anti-VDR and anti-p85 (PI 3-kinase) immunoprecipitates of D₃-treated cells were subjected to SDS-PAGE and Western blotting. Blots were probed with mAb to p85 and developed by ECL. The results shown in Fig. 7 C indicate that Abs to p85 reacted in anti-VDR immunoprecipitates with a band that presumably corresponds to the p85 subunit of PI 3-kinase. This association was only observed in cells treated with hormone, and not in untreated cells. Taken together, the findings suggest that D₃ treatment induces the formation of a signaling complex containing the VDR and PI 3-kinase. This results in activation of the lipid kinase, and is required for monocyte differentiation and induction of CD14 expression.

The steroid hormone vitamin D₃ is known to induce immature, myeloid precursor cells to differentiate into mature monocytic cells. This process is accompanied by high-level expression of mRNA and protein for CD14, and other markers such as CD11b 27,28,29. How these D₃-initiated events are regulated is not completely understood. Cellular responses to D₃ have primarily been attributed to activation of the VDR, which acts as a transcription factor modulating the expression of a variety of genes 53. In contrast to genomic signaling, several reports have implicated alternative, nongenomic mechanisms of action for D₃ involving initiation of signaling at the cell membrane 54,55,56. The objective of this study was to examine signaling events in response to D₃ in order to define further how this hormone regulates myeloid cell differentiation.

The principal conclusions drawn from the experiments reported are that D₃-induced expression of CD14 and CD11b requires both PI 3-kinase and the VDR. Moreover, this process appears to involve the formation of a PI 3-kinase–VDR signaling complex. The conclusion that D₃-induced CD14 expression is PI 3-kinase dependent is based on several lines of evidence, including: (a) in vitro kinase assays with immunoprecipitated PI 3-kinase, showing that incubation of cells with D₃ leads to activation of the enzyme (Fig. 2); (b) D₃-induced CD14 expression is abrogated in THP-1 cells incubated with the PI 3-kinase inhibitors, wortmannin and LY294002 (Fig. 3); (c) an antisense strategy to downregulate PI 3-kinase also attenuated D₃-induced CD14 expression (Fig. 4); and (d) expression of a dominant negative mutant of PI 3-kinase (Δp85) in U937 cells also completely abrogated D₃-induced expression of both CD14 mRNA and protein (Fig. 5). Similarly, CD11b induction in response to D₃ was also attenuated by wortmannin and LY294002 in both THP-1 cells and in human peripheral blood monocytes. Taken together, these findings establish that D₃-induced CD14 and CD11b expression are regulated by PI 3-kinase, and that they are consistent with a nongenomic mechanism of hormone action.

Expression of the Cdk inhibitor p21 is induced by D₃, and this is associated with the differentiation of immature myeloid cells, including the expression of CD14 52. Therefore, it was possible that inhibition of PI 3-kinase may have abrogated hormone-induced expression of p21 proximally, leading to secondary, indirect effects on the expression of CD14 and CD11b. However, the findings that neither wortmannin nor LY294002 inhibited the induction of p21 while at the same time they inhibited the expression of CD14 and CD11b (Fig. 3, Table and Table) indicated that this was not the case. These findings suggest that PI 3-kinase selectively regulates these monocyte differentiation markers independent of any effects on p21. They also suggest that D₃-induced signaling initiated through the VDR appears to involve at least two separate pathways, one leading to p21 induction that is independent of PI 3-kinase, and a second pathway that is PI 3-kinase dependent and which regulates at least the induction of CD14 and CD11b. Moreover, although p21 has been shown to be involved in regulating monocyte differentiation 52, results from the present study suggest that it is not sufficient by itself for induction of monocyte differentiation in response to D₃, and that other, PI 3-kinase– and VDR-regulated elements are required.

Induction of CD14 expression in response to D₃ is regulated at the level of gene transcription 28,29 and the CD14 promoter, particularly from bp −128 to −70, has been shown to be critical for CD14 expression in response to D₃ 28. However, this region does not contain a canonical VDRE 28. Rather, the CD14 promoter contains two GC boxes that bind the nonreceptor, transcription factor Sp1, and this interaction is believed to be essential for CD14 expression 28,57. In light of this molecular data, activation of CD14 transcription would not be expected to be a direct response to D₃ signaling through the VDR. Nevertheless, experiments that used VDR antisense S-oligo to downregulate expression of the endogenous VDR before D₃ stimulation showed that the VDR is required for D₃-induced expression of CD14 mRNA (Fig. 6). The obligate requirement for the VDR in this response was also supported by the finding that activation of PI 3-kinase in response to D₃ was markedly attenuated in cells treated with VDR antisense (Fig. 7 A). Moreover, a signaling complex containing both the VDR and PI 3-kinase was identified in D₃-treated cells (Fig. 7b and Fig. c), suggesting that a functional cytosolic VDR is a prerequisite for PI 3-kinase activation in response to D₃. These findings appear to identify a novel, nongenomic mechanism of action for the VDR. It is of interest to note that the hormone-induced formation of a VDR–PI 3-kinase signaling complex is reminiscent of the finding that activation of the Raf-MAP kinase pathway by D₃ in keratinocytes involves an association of the VDR with the adaptor protein p66^shc 58. Together, these findings suggest that other models of nongenomic signaling involving the VDR may yet be identified.

The observations that PI 3-kinase regulates CD14 expression in response to D₃ and that the VDR is directly involved in this process do not exclude the possibility of a component of genomic action by the VDR, despite the absence of a canonical VDRE within the CD14 promoter. One possibility to consider is that upon incubation of cells with D₃, the VDR may translocate to the nucleus towards D₃-responsive elements, and enhance the activity of the known CD14 transcription factor Sp1. Two observations lend support to this hypothesis: (a) the finding of transcriptional synergism between the VDR and Sp1 using a construct of the VDRE, the binding sites for Sp1, and a luciferase reporter gene 59; and (b) the presence of a VDRE-like sequence at the distal Sp1 site within the CD14 gene 60.

The findings that D₃-induced monocyte differentiation for CD14 and CD11b expression requires PI 3-kinase are consistent with previous reports in which this lipid kinase has been implicated in regulating the differentiation of various cell types, including immature myeloid cells 49,61,62,63. For example, indirect data, based solely on the use of inhibitors, suggested a potential role for PI 3-kinase in regulating the differentiation of the myeloid leukemia cells HL-60 63. In addition, when FDC-P1 myeloid cells that express the M-CSF receptor c-fms were treated with M-CSF, there occurred the rapid formation of a complex between c-fms and several signal transduction proteins, including PI 3-kinase 49. Other studies have suggested that the differentiation of FDC-P1 cells is regulated by the activities of both phospholipase C-γ and PI 3-kinase in response to M-CSF stimulation 61. In addition, stimulation of type III receptor tyrosine kinases in the same cell line was observed to lead to PI 3-kinase–dependent monocytic differentiation 62. Thus, the steroid hormone D₃ appears to share with M-CSF the property of PI 3-kinase activation during the induction of monocyte differentiation.

It is of interest to compare D₃ with phorbol esters that also act to promote monocyte differentiation. For example, PMA induces the expression of the monocyte differentiation markers CR3 (CD11b/CD18) and p150,95 64, and modulates several functional properties of myeloid precursor cells, such as intracellular adhesion molecule 1–dependent adhesion 22, phagocytosis 44,65, and bactericidal activity 66. However, PMA does not activate PI 3-kinase (Hmama, Z., and N. Reiner, unpublished data), and PMA-induced cell differentiation is resistant to PI 3-kinase inhibition 22. Consistent with the findings presented above, although low concentrations of D₃ induce an ∼50-fold increase in CD14 surface expression, optimal doses of PMA resulted in only marginal increases (Hmama, Z., and N. Reiner, unpublished data). Regarding signaling mechanisms regulating PMA-induced cell differentiation, several reports have implicated the protein kinase C–Raf-MAP kinase pathway 33,67,68. MAP kinase activity has been also shown to be activated by D₃ in keratinocytes, enterocytes, and in the promyelocytic cell lines HL60 and NB4 34,58,69. To examine whether the MAP kinase pathway may be involved in THP-1 cell differentiation, D₃-treated cells were examined for tyrosine phosphorylation of p42 and p44 MAP kinase isoforms, and assayed for MAP kinase activity using myelin basic protein as substrate. The results obtained (data not shown) indicated that MAP kinase is not activated in response to D₃ in THP-1 cells.

The mechanism by which PI 3-kinase becomes activated in a complex with the VDR is not presently known. At least one well-established mechanism for activation of PI 3-kinase is known to involve interactions of the Src homology 2 domain of the p85 regulatory subunit with tyrosine phosphorylated proteins, including both receptor- and nonreceptor-protein tyrosine kinases 35. In the VDR, the only potential sites of phosphorylation correspond to Ser⁵¹ 70 and Ser²⁰⁸ 71, and the relevance of these residues in VDR-mediated D₃ signal transduction is unknown. Thus, it is not presently clear how a direct interaction of the VDR with the p85 subunit may lead to activation of PI 3-kinase. On the other hand, a multimolecular complex involving the VDR and a tyrosine-phosphorylated adaptor protein could potentially be involved in D₃-induced activation of PI 3-kinase.

At present, it is not clear how VDR-activated PI 3-kinase would be targeted to the membrane compartment to mediate cellular responses, given the cytosolic and nuclear localization of the VDR. One possibility to consider is that there is a small, membrane-associated population of VDR molecules that induces translocation of PI 3-kinase to the membrane compartment. A second possibility is that once activated, the kinase may simply diffuse to the vicinity of the inner leaflet of the plasma membrane. Third, it is also possible that VDR-activated PI 3-kinase may act on nonmembrane-associated substrates to mediate its effects.

During the course of these studies, it was observed that IGF-I failed to induce CD14 surface expression (Fig. 1 C), despite it ability to activate PI 3-kinase (Fig. 2). Therefore, PI 3-kinase activation in response to D₃ appears to be necessary, but not sufficient to bring about CD14 expression. These findings suggest the possibility that induction of CD14 in response to D₃ may involve VDR-mediated signals that bifurcate to involve nongenomic effects of PI 3-kinase, perhaps acting in concert with a genomic mechanism of action. In this respect, cross-talk between nuclear receptor–mediated signaling and nongenomic actions have been described in D₃-treated osteoblasts 72.

In summary, the findings presented demonstrate a novel pathway in which D₃ signaling for myeloid cell differentiation involves PI 3-kinase activation and signal complex formation with the VDR, which is itself shown to be required for cell differentiation. This pathway is essential for D₃-induced expression of CD14 and CD11b, and attributes important functional roles to PI 3-kinase and the VDR in monocyte differentiation.

We thank Dr. W.C. Van Voorhis for antibody to CD14, and Mr. Raymond Lo for technical assistance.

This work was supported by grant MT-8633 from the Medical Research Council of Canada.