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Endocrinology Vol. 140, No. 10 4779-4788
Copyright © 1999 by The Endocrine Society

ARTICLES

Vitamin D Analogs, 20-Epi-22-Oxa-24a,26a,27a,-Trihomo-1{alpha},25(OH)2-Vitamin D3, 1,24(OH)2-22-Ene-24-Cyclopropyl-Vitamin D3 and 1{alpha},25(OH)2-Lumisterol3 Prime NB4 Leukemia Cells for Monocytic Differentiation via Nongenomic Signaling Pathways, Involving Calcium and Calpain1

Donna M. Berry and Kelly A. Meckling-Gill

Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada, N1G 2W1

Address all correspondence and requests for reprints to: Kelly A. Meckling-Gill, Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada, N1G 2W1. E-mail: kmeckling.ns{at}APS.UoGuelph.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Side-chain modified vitamin D analogs including 20-Epi-22-oxa-24a,26a,27a-trihomo-1{alpha},25-dihydroxyvitamin D3 (KH1060), and 1,24-dihydroxy-22-ene-24-cyclopropyl-vitamin D3 (MC903) were originally designed to aid in the treatment of hyperproliferative disorders including psoriasis and cancer. Here we demonstrate that these analogs, as well as the 6-cis-locked conformer, 1{alpha},25-dihydroxy-lumisterol3 (JN) prime NB4 cells for monocytic differentiation. Previously, the action of MC903 and KH1060 was presumed to be mediated by the nuclear vitamin D receptor (VDRnuc). Differentiation in response to all analogs was shown to be inhibited by 1ß,25-dihydroxyvitamin D3 (HL), the antagonist to the nongenomic activities of 1,25D3. These data suggest that although MC903 and KH1060 may bind the VDRnuc, that the differentiative activities of these agents requires nongenomic signaling pathways. Here we show that 1{alpha},25(OH)2-d5-previtamin D3 (HF), JN, KH1060, and MC903 induce expression of PKC{alpha} and PKC{delta} and translocation of both isoforms to the particulate fraction, and PKC{alpha} to the nuclear fraction. The full differentiation response with combinations of analogs and TPA was inhibited 50% by the mem-brane permeable Ca2+ chelator, 1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) or calpain inhibitor I. These data demonstrate that intracellular free calcium and the calcium-dependent protease, calpain play critical roles in monocytic differentiation. Intracellular calcium appears to be most critical in the 1,25D3-priming stage of differentiation, while calpain is essential in the TPA maturation response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ACTIVE FORM of vitamin D, 1{alpha},25dihydroxyvitamin D3 (1,25D3), and analogs of 1,25D3 have been shown to inhibit the growth of cancer cell lines including breast (1, 2, 3, 4), colon (5, 6, 7), prostate (1, 8, 9, 10), and leukemia cells (1, 11, 12, 13, 14). Mechanisms of growth inhibition may include induction of apoptosis or promotion of differentiation (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14). Until recently, much of the activity of 1,25D3 had be attributed to the induction of signaling pathways dependent on the binding of 1,25D3 to the nuclear vitamin D receptor (VDRnuc) (review Ref. 15). More recently 1,25D3 has been shown to act through VDRnuc-independent signaling pathways, including changes in intracellular calcium levels (16, 17), rapid effects on phospholipid metabolism (18, 19), and the direct activation of protein kinase C (PKC) (20, 21). To aid in the study of the VDRnuc-independent effects of 1,25D3, analogs of 1,25D3 that can activate nongenomic pathways, but which have very low affinity to the VDRnuc have been synthesized (22, 23, 24). There is some evidence for a second receptor for 1,25D3 that is associated with the plasma membrane and is responsible for the rapid, nongenomic effects of 1,25D3 (18, 21, 25, 26).

The NB4 cell line was derived from a patient with acute promyelocytic leukemia (APL) (27), and contains the characteristic 15; 17 translocation (27, 28). Studies have shown this cell line to be bipotent, with potential to differentiate into neutrophils or monocytes in response to all-trans-retinoic acid (27) or 1,25D3 plus 12-O-tetradecanoylphorbol-13-acetate (TPA), respectively (14). Differentiation therapy using naturally occurring retinoic acid has shown some success clinically (29) however, one difficulty in applying 1,25D3 to clinical trials is its relatively high toxicity due to the induction of hypercalcemia in vivo (30). Noncalcemic analogs of 1,25D3, which are effective inducers of monocytic differentiation may be safer in a clinical setting. An understanding of the molecular mechanisms by which 1,25D3 and TPA act should provide insight into the appropriate design of improved differentiation therapies for leukemia. This information will also increase our understanding of the signaling pathways involved in normal monocyte/macrophage development.

Previous studies in our laboratory have shown that NB4 cells, unlike the myelocytic cell line HL-60, require both 1,25 D3 and TPA to undergo monocytic differentiation (14). In addition, we have shown that PKC activity is necessary for monocytic differentiation (31) and that 1,25 D3 and a nongenomic analog 1{alpha},25-dihydroxy-9,14,19,19,19-pentadeuterio-previtamin-D3 (HF), induce an up-regulation of both PKC{alpha} and PKC{delta} and cause the translocation of both these isoforms to the nuclear and membrane fractions of NB4 cells (32). The 1{alpha},25-dihydroxyvitamin D3 molecule exhibits conformational flexibility, existing as either 6-cis or 6-trans conformers. Recent studies from the lab of Norman have shown evidence that the VDRnuc and VDRmem are preferentially activated by analogs in the 6-trans and 6-cis conformations, respectively (24). The nongenomic analogs HF and 1{alpha},25-dihydroxy-lumisterol3 (JN) are locked in the 6-cis conformation, and as such have very low affinity for the VDRnuc. It has been shown in previous studies that HF and JN activate the membrane-associated signaling pathways and have very little effect on genomic pathways involving the VDRnuc (22, 23) The nongenomic antagonist, 1ß,25dihydroxyvitamin D3 (HL) (33), can inhibit both the monocytic differentiation of NB4 cells (34) and the 1,25D3-induced effects on PKC translocation (32). These studies suggest that activities of 1,25D3, which are not dependent on the VDRnuc, are important for monocytic differentiation of NB4 cells.

In this present study we examined monocytic differentiation of NB4 cells in response to TPA combined with 1,25D3 or one of four analogs, each with a different calcemic index and affinity for the VDRnuc (see Table 1Go). Induction of monocytic differentiation in response to all these analogs is mediated by VDRnuc-independent signaling pathways involving intracellular calcium and the calcium-dependent protease, calpain, activity. All four analogs induce an up-regulation and membrane-translocation of both PKC{alpha} and {delta}, and translocation of the {alpha} isoform to the nucleus.


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  		Table 1. Characteristics of analogs

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
NB4 cells were obtained from the laboratory of H. Gronemeyer (Strasbourg, France). NB4 cells were originally isolated from bone marrow cells of an acute promyelocytic leukemia patient in relapse. Cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% FCS (Life Technologies, Inc.) and 50 U/ml of penicillin and streptomycin. An initial mycoplasma infection with M. Arginini was treated with BM cyclin (Roche Molecular Biochemicals) and cells were subsequently screened for mycoplasma on a routine basis. Cells were maintained at 37 C in a humidified atmosphere containing 5% CO2 in tissue culture flasks (7–22ml, Corning, Inc.). Stock cultures were maintained in log phase of growth and passaged every 2 days. Cell densities were determined using a Coulter Counter (model ZM).

Reagents
1{alpha},25-dihydroxyvitamin D3(1,25D3), 20-Epi-22-oxa-24a,26a,27a-trihomo-1{alpha},25-dihydroxyvitamin D3 (KH1060), and 1,24-dihydroxy-22-ene-24-cyclopropyl-vitamin D3 (MC903) were generous gifts of Dr. Laurence Fraher (St. Joseph’s Health Centre, London, Ontario, Canada). 1ß,25-dihydroxyvitamin D3 (HL), 1{alpha},25-dihydroxy-lumisterol3 (JN) and 1{alpha},25-dihydroxy-9,14,19,19,19-pentadeuterio-previtamin-D3 (HF) were generous gifts of Dr. Anthony Norman (University of California, Riverside, CA). All analogs of 1,25D3 were dissolved in ethanol to give stock solutions ranging from 10-6 M to 10-4 M, and stored under nitrogen at -20 to -80 C. The cell permeable acetoxymethyl (AM) ester derivative of 1,2-bis(o-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid (BAPTA) was purchased from Sigma, dissolved in dimethylsulfoxide (DMSO) and stored at -20 C for no more than 4 weeks. 12-O-tetradecanoylphorbol-13-acetate (TPA) was purchased from Sigma, dissolved in ethanol and stored at -20 C. Calpain inhibitor I was purchased from Roche Molecular Biochemicals, dissolved in ethanol and stored at -20 C.

Treatment of cells
For differentiation assays NB4 cells were seeded at 2.5 x 105 cells/ml in 6-well plates, primed with 1,25D3 or analog for 8 h, then treated with TPA (200 nM) for 64 h, for a total treatment time of 72 h. For the phase studies (Fig. 7Go), cells were primed with 200 nM 1,25D3 with or without BAPTA (1 µM) or calpain inhibitor (2 µM) for 8 h, washed three times with PBS, incubated in drug-free media for 30 min and then suspended in 200 nM TPA (± BAPTA or calpain inhibitor) for an additional 64 h.



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  		Figure 7. Treatment of NB4 cells with BAPTA and calpain inhibitor in the 1,25D3-priming phase (phase I) and TPA phase (phase II), respectively, leads to optimal inhibition of monocytic differentiation in response to 1,25D3 and TPA. Cells (2.5 x 105) were treated in two separate phases; 8 h of treatment with 50 nM 1,25D3, followed by 64 h of treatment with 200 nM TPA. The phases were separated by three washes in PBS and a 30-min period of incubation in reagent-free media. BAPTA (1 µM) or calpain inhibitor (2 µM) were introduced 30 min before phase I, II, or both phases to determine which phases of differentiation required intracellular calcium or calpain activity. Adherence, CD14 expression and esterase expression were measured as described in Materials and Methods section, and used as criteria for induction of monocytic differentiation. Shown are averages of three separate experiments. Bars not sharing a letter are statistically different (P < 0.05, n = 3).

 
Measurement of adherence
The nonadherent population of cells was prepared by combining the suspended cells with those collected after a gentle wash of PBS. The adherent cells were then incubated with cold PBS containing 10 mM trypsin-EDTA for 10 min, lifted by a series of gentle washes, and collected. This process was then repeated.

Cells were quantified with a Coulter Counter (Model ZM). Adherence was calculated and expressed as a percentage of total cells in each treatment.

CD14 assay
After each treatment the percentage of cells expressing CD14 surface antigen was determined using magnetic polystyrene beads (Dyanbeads, Lake Success, NY) as we have previously described (31). In brief, cells were washed three times with PBS and 2 x 105 cells were resuspended in PBS + 2% FBS with approximately 10 beads/cell in a microcentrifuge tube. The cells were incubated with the beads at 4 C on an orbital shaker for 2 h. Cells were then exposed to a magnet for 2 min and the remaining CD14 negative cells were counted. The number of cells that were CD14 positive were calculated by difference and expressed as a percentage of total cell number.

Nonspecific esterase assay
After each treatment, the percentage of cells expressing nonspecific esterase activity was determined using a kit purchased from Sigma as previously described (14). The assay was carried out in 12-well plates and cells expressing esterase activity (with black granules) were counted in a blinded fashion, with at least 250 cells counted in each treatment group.

Cellular fractionation
Samples of cellular fractions were prepared using a previously described fractionation method (32) with a few modifications. Cells were collected after 12 h of treatment, washed three times in ice-cold PBS + complete protease inhibitors (Roche Molecular Biochemicals, catalog no. 1697498), a small aliquot was removed for whole cell sample and the remaining cells were resuspended in hypotonic lysis buffer (20 mM Tris-HCl, 5 mM EGTA + protease inhibitors) and chilled on ice for 10 min. Cells were then disrupted by Dounce homogenization, and the nuclear fraction was collected in a 10 min centrifugation (500 x g). The supernatant from the 500 x g spin was then centrifuged at 100,000 x g to collect the particulate fraction. The cytosolic proteins were precipitated from the supernatant of the 100,000 x g spin with 10% TCA (30 min, 4 C). The precipitated proteins were solubilized by resuspending in hot 5M urea. A 5'-nucleotidase(5'-ND) assay (Sigma) was performed to determine the degree of plasma membrane enrichment of the particulate fraction. Results of that assay showed that 5'-ND activity was approximately 5 or 22 times higher in the particulate fraction compared with the whole or nuclear fractions, respectively (data not shown). A protein assay was performed on all samples by the method of Bradford (35) using a kit from Bio-Rad Laboratories, Inc. (Hercules, CA). The fractions were resuspended in Laemmli buffer with protease inhibitors and stored at -20 C for subsequent analysis.

Western blotting
Proteins were separated by SDS-PAGE on 12% acrylamide gels and transferred to polyvinylidene difluoride membrane (Gelman) on a semidry transfer apparatus 2 h at 200 mA. Gels were loaded on the basis on protein quantity and equal loading was determined by staining the membranes with Fast Green. Membranes were blocked with 5% milk powder in Tris-buffered saline (TBS), washed with TBS with 0.05% Tween-20 (TBS-T), and incubated with primary antibody for 1 h at room temp (RT). The primary murine monoclonal antibody used to detect calpain I was a generous gift from Dr. John Elce (Queen’s University, Kingston, Ontario, Canada). The monoclonal antibodies used to detect PKC{alpha} and PKC{delta} were purchased from Transduction Laboratories (Lexington, KY). Antibody dilutions used for PKC{alpha}, PKC{delta}, and calpain were 1:1,000, 1:1,000, and 1:2000, respectively. Membranes were then washed with TBS-T for 30 min and incubated for 1 h at RT with secondary antibodies conjugated to horseradish peroxidase. The secondary antibody used for all blots was antimouse IgG at a concentration of 1:25,000 (Sigma) in TBS-T with 5% milk powder. Polypeptides were detected using the ECL kit (Amersham Pharmacia Biotech) and quantified by autoradiography and densitometry.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
1,25D3, HF, JN, KH1060 and MC903 induce monocytic differentiation when combined with TPA; differences in potency do not correlate with affinity to VDRnuc
Markers of monocytic differentiation in response to 8 h priming with each analog, followed by treatment with 200 nM TPA, were assessed after a total of 72 h (Fig. 1Go). Markers included measurement of adherence, CD14 expression and expression of nonspecific esterase. Treatment with 1,25D3, HF, JN, KH1060, or MC903 in the absence of TPA did not induce differentiation (data not shown). Dose/response curves were generated for each analog to determine the approximate minimum concentration required for maximal differentiation when combined with TPA. Potency was as follows: KH1060 (1 nM) > HF (10 nM) > MC903 (150 nM) > 1,25D3 (200 nM) {approx} JN (200 nM). This order of effectiveness does not correlate with the affinity of each analog for the VDRnuc, as previously determined (15): 1,25D3 {approx} MC903 {approx} KH1060 > HF > JN.



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  		Figure 1. Monocytic differentiation in response to analogs combined with 200 nM TPA. Cells (2.5 x 105) were treated with each analog for a period of 8 h, followed by treatment with 200 nM TPA for 64 h. The degree of differentiation was determined by measuring adherence, CD14 expression and esterase expression as described in Materials and Methods section. a–e, Optimum concentrations of 1,25D3, HF, JN, KH1060, and MC903, are 200 nM, 10 nM, 200 nM, 1 nM, and 150 nM, respectively. Shown are averages of three separate experiments. Columns not sharing a letter are statistically different (P < 0.05, n = 3).

 
Monocytic differentiation in response to 1,25D3 and analogs involves nongenomic signaling pathways
To determine if nongenomic responses to the analogs were necessary to induce differentiation, the nongenomic antagonist, 1ß,25dihydroxyvitamin D3 (HL) was used. Exposure to this antagonist (100 nM) during the process of differentiation resulted in a substantial reduction in three markers of differentiation in response to all of the analogs combined with 200 nM TPA. The addition of HL to the combination of TPA and one each of 200 nM 1,25D3, 10 nM HF, 200 nM JN, 1 nM KH1060, and 150 nM MC903, reduced expression of differentiation markers nearly to the levels observed with TPA alone (Fig. 2Go).



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  		Figure 2. The nongenomic antagonist HL, inhibits differentiation of cells treated with 1,25D3, HF, JN, KH1060, or MC903 combined with TPA. Cells (2.5 x 105/ml) were treated with 200 nM 1,25D3(D3), 10 nM HF(HF), 200 nM JN(JN), 1 nM KH1060(KH), or 150 nM MC903(MC) combined with 100 nM HL or vehicle for 8 h. Following the 8 h-priming period, all analog or 1,25D3-treated cells were treated with 200 nM TPA for 64 h. Adherence, CD14 expression, and esterase expression were measured as markers of differentiation, as described in the Materials and Methods section. Data shown are averages of three separate experiments. Bars not sharing a letter are statistically different (P < 0.05, n = 3).

 
Monocytic differentiation in response to all analogs involves intracellular calcium and calpain activity
Calpain I inhibitor and BAPTA were used to determine if calcium-dependent protease (calpain) activity or intracellular calcium were necessary for the monocytic differentiation of NB4 cells (Fig. 3Go) Suboptimal concentrations of 1,25D3 and analogs were used in this particular experiment to allow low, nontoxic concentrations of BAPTA and calpain inhibitor to be used. A toxicity assay was performed on the NB4 cells to determine the maximum concentration of BAPTA and of calpain inhibitor, which did not cause growth arrest or induce death in the cells. Results showed that 1 µM BAPTA and 2 µM calpain inhibitor were the highest concentrations which could be tolerated by the cells (data not shown); these concentrations are much lower than those used in other studies (36, 37). Chelation of intracellular calcium by 1 µM BAPTA inhibited differentiation by approximately 50%, based on adherence, CD14 and esterase expression (Fig. 3Go). Similar results were found when 2 µM calpain inhibitor was present in the media, inhibiting differentiation by approximately 50% in response to TPA combined with each of the analogs (Fig. 3Go).



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  		Figure 3. BAPTA and calpain inhibitor I, inhibit differentiation in response to 1,25D3 or analogs combined with TPA. BAPTA or calpain inhibitor (1 µM, or 2 µM) were introduced into the cell cultures 30 min before treatment with analogs and TPA began. Cells (2.5 x 105/ml) were primed with suboptimal concentrations of 1,25D3 or analogs (50 nM 1,25D3, 1 nM HF, 100 nM JN, 0.1 nM KH1060, or 10 nM MC903) for 8 h and then treated with 200 nM TPA. Markers of differentiation were measured at the end of the 72.5 h treatment period as described in Materials and Methods section. Data shown are averages of three separate experiments. *, Statistical difference from the same treatment without chelator or inhibitor (P < 0.05, n = 3).

 
Expression of calpain is increased in response to treatment with 1,25D3
A previous study showed that 1,25D3 treatment increases the expression of calpain I in renal carcinoma cells (38). To determine if 1,25D3 had effects on the expression of calpain I protein in our model, Western blot analysis was performed. Figure 4aGo shows a time course of calpain I expression after treatment with 200 nM 1,25D3. This time period correlates with the time NB4 cells need to be primed with 1,25D3 to induce a maximal maturation response to TPA (31). The increase in calpain expression after 8 h of 1,25D3 treatment is blocked by addition of HL to the culture media (Fig. 4bGo), demonstrating that nongenomic signaling pathways are required for this response to 1,25D3.



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  		Figure 4. Calpain I expression is up-regulated in response to 1,25D3. Expression of calpain I protein was determined at 0, 0.5, 1, 2, 4, and 8 h after treatment with 200 nM 1,25D3 (a) or 200 nM 1,25D3 and 100 nM HL (b). Whole cell lysates were extracted and Western blot analysis was performed as described in Materials and Methods section. A total of 4.0 x 105 cells were load per lane (to obtain detection within the linear range), and equal loading of lanes was assessed by staining membrane with Fast Green. Autoradiographs were developed after 5–10 min of exposure, using ECL detection. Shown are representative blots of at least three separate experiments.

 
PKC{alpha} and PKC{delta} are up-regulated and translocated in response to 1,25D3, HF, JN, KH1060 and MC903
PKC{alpha} and PKC{delta} expression were examined after 12 h of treatment with 200 nM 1,25D3, 10 nM HF, 200 nM JN, 1 nM KH1060, or 150 nM MC903. Figure 5aGo shows the expression of PKC{alpha} in whole cell lysates and the three cellular compartments that were isolated. The expression of PKC{alpha} is increased in the whole cell lysates after 12 h of treatment with 1,25D3 and analogs, when compared with vehicle-treated cells (Fig. 5aGo). In the nuclear compartment, PKC{alpha} is not detectable in cells treated with vehicle but is translocated to this compartment in response to 1,25D3 or each of the analogs (Fig. 5bGo). Similarly, there is an increased expression of PKC{alpha} in the particulate compartment of the cells treated with 1,25D3 or analog for 12 h (Fig. 5cGo). Figure 5dGo shows that there is little change in the expression of the isoform in the cytosolic compartment.



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  		Figure 5. PKC{alpha} is translocated to the nuclear and particulate cellular fractions and total expression is up-regulated in response to 1,25D3, HF, JN, KH1060, or MC903. NB4 cells (2.5 x 105/ml) were treated with 200 nM 1,25D3, 10 nM HF, 200 nM JN, 1 nM KH1060, or 150 nM MC903 for 12 h, after which cellular fractionation and Western blotting were performed as described in Materials and Methods section. Equal quantities of protein were loaded in each lane. Forty micrograms of the whole cell lysate and nuclear preparations, and 20 µg of the particulate and cytosolic preparations were loaded. Fast Green staining of membranes was used to assess equal loading of lanes. Blots were exposed to film for 5 min–2 h to achieve densities within the linear range. Shown are blots representative of at least three separate experiments.

 
The expression of PKC{delta} was similar to that observed for the {alpha} isoform. When NB4 cells were treated with 1,25D3 or analogs, PKC{delta} expression increased in whole cell lysates (Fig. 6aGo) and was translocated to the particulate cell fraction within 12 h (Fig. 6bGo). Unlike the {alpha} isoform, PKC{delta} expression was not increased in the nuclear fraction after 12 h of treatment; however, we have previously demonstrated nuclear association with 1,25D3 and HF after 24 h of treatment (32). Expression of PKC{delta} did not appear to be lower in the cytosolic fractions of 1,25D3 or analog-treated cells compared with treatment with vehicle alone (Fig. 6cGo).



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  		Figure 6. PKC{delta} is translocated to the particulate cellular fraction and total expression is up-regulated in response to 1,25D3, HF, JN, KH1060, or MC903. NB4 cells (2.5 x 105/ml) were treated with 200 nM 1,25D3, 10 nM HF, 200 nM JN, 1 nM KH1060, or 150 nM MC903 for 12 h, after which cellular fractionation and Western blotting were performed as described in Materials and Methods section. Equal quantities of protein were loaded in each lane. Forty micrograms of the whole cell lysate and nuclear preparations, and 20 µg of the particulate and cytosolic samples were loaded. Fast Green staining of membranes was used to assess equal loading of lanes. Blots were exposed to film for 5 min–2 h to achieve densities within the linear range. Shown are blots representative of at least three separate experiments.

 
Intracellular calcium is required for the 1,25D3-priming stage of differentiation, while calpain activity is required during the TPA-phase of maturation
Figure 7Go shows the results of an experiment conducted to determine which phase of the differentiation was affected by chelation of calcium and inhibition of calpain activity. All treatment groups were exposed to 50 nM 1,25D3 for 8 h (phase I, priming) and then exposed to 200 nM TPA for the remainder of the 72-h treatment period (phase II, maturation). BAPTA (1 µM) or calpain inhibitor (2 µM) were added to the media in phase I, phase II, or both phases. The cells were incubated in media with the chelator or inhibitor for 30 min before treatment with 1,25D3 or TPA to allow them to accumulate in the cells. The results indicate that BAPTA has its largest inhibitory effect when used in phase I of differentiation, and results were not significantly different when the chelator was applied in both phases. Exposing the cells to BAPTA in phase II caused a significant inhibition of differentiation when compared with control, but the effect was significantly lower than when cells were exposed to BAPTA in phase I. Inhibition of calpain had the largest effect on differentiation when cells were exposed to the inhibitor in phase II, and results were not significantly different when the cells were exposed to calpain inhibitor during both phases. Calpain inhibitor in phase I also caused a significant decrease in the markers of differentiation when compared with control cells, but the inhibition was significantly lower than inhibition caused by calpain inhibition in phase II.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The NB4 cell line requires the synergy of 1,25D3 and phorbol ester to undergo monocytic differentiation. Many leukemia cells lines of different origins can be induced to undergo monocytic differentiation in response to either of these reagents alone (11, 39, 40, 41). A previous report from our lab showed that the combination of 1,25D3 and TPA led to the greatest induction of differentiation when NB4 cells were exposed to 1,25D3 before TPA, suggesting that 1,25D3 primed the cells to respond to TPA (31). It is still currently unknown how 1,25D3 primes NB4 cells for monocytic differentiation. In the present study we show that in addition to 1,25D3 and the previously tested analog HF, JN, KH1060, and MC903 also prime NB4 cells for monocytic differentiation in response to TPA. The priming effect of 1,25D3 and all of the analogs tested, appears to be mediated through the regulation of PKC{alpha}, PKC{delta}, and the neutral, calcium-activated protease, calpain.

It has been shown in previous studies that analogs of 1,25D3, can induce monocytic differentiation in leukemia cells lines other than NB4 with a greater efficiency than 1,25D3 itself (13, 42, 43, 44). The increased efficiency of these analogs cannot be explained by increased affinity to the VDRnuc, due to the fact that many of the analogs studied have lower affinities to the VDRnuc when compared with 1,25D3 (13, 42, 43, 44). It should be noted that most of the affinity assays were performed using VDRnuc protein isolated from chick intestine, which may differ from VDRnuc isolated from leukemia cells (15). Nevertheless, we provide further evidence that pathways other than those mediated by VDRnuc are critical to the activities of many vitamin D3 analogs. For example we show that KH1060, an 20-epi analog with affinity to the VDRnuc similar to that of 1,25D3 (45), can induce differentiation equivalent to 1,25D3 at concentrations 200-fold lower. Other studies have shown that 20-epi analogs of 1,25D3, including KH1060, can induce conformational changes in the VDRnuc, thereby achieving greater transcriptional activity when compared with 1,25D3, despite similar affinities to the VDRnuc protein (46, 47). However, an hypothesis that binding to the nuclear receptor is not the primary factor in monocytic differentiation of NB4 cells is supported by the fact that the 6-cis-locked analogs, JN and HF, can induce differentiation quite effectively when combined with TPA, and these analogs have very low affinity for the VDRnuc (22, 23). In the case of HF, its affinity to the VDRnuc is 10-fold lower than 1,25D3, but it can induce differentiation in NB4 cells at concentrations approximately 20-fold lower than 1,25D3 (34). Clearly binding to the VDRnuc does not determine the relative effectiveness of the analogs. While VDRnuc could mediate some of the differentiating activity of analogs such as KH1060 and MC903, the observation that differentiation can be almost completely inhibited by interfering with the rapid, nongenomic effects of 1,25D3 and analogs, using HL, strongly implies that nongenomic signals are necessary for monocyte differentiation. One hypothesis would be that the differences in effectiveness of these analogs is due to differences in their affinity for the putative VDRmem. Until this receptor is fully characterized it will not be possible to directly test this possibility.

The use of 1,25D3 in clinical trials is often limited by its high toxicity due calcemia induction in vivo (15, 30). It is important to note that HF (22) and MC903 (48) have very low in vivo calcemic indices when compared with 1,25D3, and in the case of HF can induce differentiation at much lower concentrations than 1,25D3. In addition, while KH1060 has calcemic effects similar to that of 1,25D3, toxicity may not be a problem in vivo because it induces differentiation at concentrations 2-orders of magnitude lower than 1,25D3.

Numerous reports have indicated that the PKC family of kinases is involved in myeloid differentiation. The {alpha}, ß, and {delta} isoforms are among those shown to be regulated during the differentiation of leukemia cell lines other than NB4 (49, 50, 51). Our lab has shown in previous reports that PKC activity is necessary for induction of monocytic differentiation of NB4 cells (22). We have also shown that PKC{alpha} and PKC{delta} are regulated, both by increased expression and translocation, in response to 1,25D3 and its 6-cis analog HF (32). Evidence that this regulation of PKC required the nongenomic actions of HF was provided by showing that the nongenomic antagonist HL could inhibit the translocation and increased expression of PKC (32). In this report we show that JN, KH1060 and MC903 also regulate the expression of PKC {alpha} and {delta}, through increased expression and translocation to the membrane fraction of the cells, and in the case of the {alpha} isoform, translocation to the nucleus. We hypothesize that part of the process of priming cells for monocytic differentiation in response to 1,25D3 involves the translocation of PKC to sites in the cell where its target proteins are localized, or where it can be readily activated by TPA. It has also been shown that 1,25D3 can directly activate PKC (20), and a recently described membrane receptor for 1,25D3 could be responsible for this activation (21). A recent report from the lab of Norman has shown that MAPK, a downstream target of PKC, is regulated in response to either 1,25D3 or an analog with low affinity to the VDRnuc (JN), suggesting that 1,25D3 activates PKC in the absence of TPA, through rapid nongenomic mechanisms (52). This information, combined with the fact that 1,25D3 does not induce monocytic differentiation without TPA, suggests that activation of PKC by 1,25D3 may be necessary, but is not sufficient to induce differentiation. It could be that 1,25D3 and TPA act in synergy to induce differentiation by activating alternate isoforms of PKC, as shown in other cell models (53), or possibly by acting at different active sites on the same isoform.

The role of intracellular calcium in the differentiation of leukemia in response to 1,25D3 has been well documented (54, 55, 56). It has also been shown that calpain, a neutral calcium-dependent protease is involved in differentiation of muscle cells and inhibition of calpain activity has been shown to limit differentiation of these cells (57). Until our report, no one had shown that calcium and calpain were important in the monocytic differentiation of NB4 cells. We show here for the first time that BAPTA, a calcium chelator, and calpain inhibitor can inhibit differentiation in response to 1,25D3 and TPA. By using BAPTA and calpain inhibitor in the different phases of differentiation, we also show that calcium is necessary in the 1,25D3-priming of NB4 cells, and that calpain activity may be necessary in the TPA phase of differentiation. The expression of calpain increased in response to 1,25D3, a response that has also been shown to occur in renal cells (38). 1,25D3 may prime NB4 cells for maturation by TPA through release of intracellular calcium stores or by initiating the uptake of calcium from the environment, as has been shown in HL-60 cells (56). It has been shown in other studies that calpain is responsive to TPA (58, reviewed in Ref. 59). 1,25D3 could prime cells for differentiation by creating a calcium-rich environment for calpain activation, or increasing the amount of calpain available for activation. Some investigators have suggested that calpain dependent proteolysis regulates PKC activity and thus controls muscle cell differentiation (60, reviewed in Ref. 59). Further study is needed to determine the exact role that calcium and calpain play in differentiation induced by 1,25D3 and TPA.

In conclusion, 1,25D3 and the analogs HF, JN, KH1060, and MC903 induce monocytic differentiation of NB4 cells when combined with TPA. Nongenomic signals including PKC{alpha} and PKC{delta}, calcium, and the protease calpain play central roles in both priming and maturation phases of this process. Further elucidation of the mechanisms by which 1,25D3 and its analogs modulate cell differentiation should aid in the design of differentiation-based therapies for promyelocytic leukemia and, possibly other neoplastic and hyperproliferative disorders.


   Footnotes
 
1 This work was supported in part by a grant from the Natural Sciences Engineering Research Council of Canada. Back

Received March 2, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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