Human PAF receptor gene expression: Induction during HL-60 cell differentiation

Vol. 181, No. 3, 1991 December 31, 1991

HUMAN

Rliane

BIOCHEMICAL

PAF RECEPTOR

Mullerl,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1580-1586

GENE EXPRESSION: CELLDIFFERENTIATION

Gilles Dupuis2,

Sylvie Turcottel

INDUCTION

and Marek

DURING

HL-60

Rola-Pleszczynskif

lhnmunology Division and 2Department of Biochemistry, Faculty of Medicine, University of Sherbrooke, Sherbrooke, QC, Canada JlH 5N4 Received

November

15,

1991

Platelet-activating factor is a potent lipid mediator of inflammation and immune regulation. Its numerous biological activities are mediated through specific receptors on the plasma membranes of responsive cells. The expression of such receptors may be modulated by various agents, including those responsible for cell differentiation. Here, we demonstrate that differentiation of the human promyelocytic leukemia cell line HL-60 by la,25(OH)2 vitamin D3 towards the macrophage phenotype is associated with induction of PAF receptor gene PAF receptor mRNA accumulation correlates with the induction and expression: development of specific PAF responsiveness as assayed by [Cag+]i fluxes. Our studies suggest that PAF responsiveness parallels macrophage differentiation and that PAF receptor expression can be regulated at the transcriptional level. 0 1991

Academic

Press,

Inc.

Platelet-activating factor (PAF) is a lipid mediator with numerous biological activities related to inflammatory and immune responses (l), as well as respiratory, cardiovascular, reproductive and nervous system physiology (2). PAF acts via receptors present on the plasma membranes of responsive cells. These receptors are stereo-specific and PAF-dependent cellular responses can be inhibited by a variety of structurally distinct PAF antagonists (2). Circumstantial evidence based on binding studies and bimodal concentration-dependent responses in target-specific cells has suggested the possibility that at least two types of PAF receptor (PAF-R) exist (3-7). Recently, Honda et aE have cloned and expressed one such PAF receptor (KD: 6.4 nM) derived from guinea pig lung. The amino acid sequence revealed that this receptor belongs to the G-protein-coupled receptor superfamily (8). Although the HL-60 promyelocytic leukemia cell line does not express significant levels of PAP-R on its surface, it can be induced to differentiate into granulocyte-like cells, with concomitant expression of specific *

To whomcorrespondence should be addressed.

0006-291X/91 Copyright All rights

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0 1991 by Academic Press, Inc. of reproduction in any form reserved.

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PAF binding sites and responsiveness to PAF (g-10). We have recently shown that HL-60 cells, induced to differentiate towards the monocyte/macrophage phenotype, progressively acquire responsiveness to nanomolar concentrations of PAF in terms of [Caz+]i mobilization and production of the cytokine tumor necrosis factor (4). It was thus of interest to determine whether the increased expression of functional cell surface PAF-R was linked to an augmented PAF-R gene message and/or a post-translational regulatory mechanism. We used the HL-60 cell line to correlate PAF-R mRNA expression with cell responsiveness to PAF during la,25(CH)2 vitamin D3 (VitDs)-dependent differentiation and report that the induction of PAF-R mRNA expression occurs during the differentiation process. MA-

Am METHODS

Cell cultHL-60 and THP-1 cells (American Type Culture Collection, Rockville MD) were maintained in Iscove’s modified Dulbecco’s and RPM1 1640 medium respectively (GIBCO Laboratories Co., Burlington Ont.), containing 10% heat-inactivated fetal bovine serum (Intergen, N.Y. NY), gentamicin (40mg/l: Schering, Pointe Claire Qc.> and 50 @I 2-mercaptoethanol for THP-1 cell cultures, in a moisture-laden atmosphere of 5% CO2 in air at 37OC. Cells were subcultured twice a week with a seeding density of 2 x 105 cells/ml. Differentiation was induced by addition of lo-7 M VitDs (41, a generous gift of Dr. M. Uskokovic (Hoffman-LaRoche, Nutley NJ). Culture conditions during VitDa stimulation were as follows: two days after passage (day 01, the cells were diluted to 3 x 10s cells/ml and VitD3 was added. After 4 days of incubation, the cells were diluted 1.5 time with fresh medium containing VitD3. K@+1;ization: Intracellular free calcium [Cag+]i was determined using the fluorescent dye Fura2-AM (Calbiochem, San Diego CA). Measurements and calibrations were performed on a SLM/Aminco spectrofluorimeter (SLM Instruments, Urbana IL) as described by Bastin et al. (11) with some modifications: undifferentiated, differentiated HL-60 or THP-1 cells were washed twice with Iscove’s or RMPI 1640 medium and suspended in Hanks’ balanced salt solutions (HBSS; GIBCO) without Ca2+ and supplemented with 35Omg/l NaHCO3 and 10 mM Hepes (pH 7.0). The concentration of Ca2+ was brought to 1.5 mM by adding a solution of CaC12 into the cuvette 10 minutes before recordings. Maximal fluorescence (Fmax) was obtained by adding Triton X-100 to a final concentration of 0.5%. Minimal fluorescence (F min) was determined by subsequent addition of EGTA, in Tris.HCl buffer (lOOmM, pH 9.0) to 125 mM. Stimuli consisted of PAF (hexadecyl analog; Bachem, Philadelphia PA), formyl-methionyl-leucinephenylalanine (FMLP; Sigma, St.Louis MO), in the absence or presence of WEB 2086 (a generous gift of Dr. H. Heuer, Boehringer-Mannheim, Mannheim FRG). PolvA+-prenaration and No thern blot analvsis; Total cellular RNA of cultured cells was isolated using therguanidinium-thiocyanate-phenol-chloroform method of Chomczynski et Sac&i (12). Subsequent purification of the PolyA+ fraction was achieved with a mRNA purification kit (Pharmacia, Baie d’Urfe Qc.). Gel electrophoresis of PolyA+ RNA (10 ug (HL-60) or 16 ug (THP-1) per lane) was performed under denaturing conditions on a 0.7% agarose gels. Transfer to a HybondN nylon membrane (Amersham, Oakville Ont.) was followed by 1581

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hybridization at 47 oC under conditions described elsewhere (13). As probe for the PAF-R, we used either the 895 bp long -1 fragment (Fig. 2A) spanning the almost entire open reading frame of the guinea pig PAF-R (8) or the whole cDNA consisting of a 3.026 kb &I--I fragment (Figs. 2B & 3B). We are grateful to Dr. T. Shimizu (University of Tokyo, Japan) for providing this clone. Control hybridizations were performed with the 1.0 kb E&I fragment of the hGAPDH gene, a kind gift of Dr. C. Asselin (University of Sherbrooke). The probes were labelled with the multiprime DNA label&g kit (Amersham) using a[32P]dCTP (specific activity >3000Ci/mmole, Amersham).

[Ca2+]i mobilization PAF triggers [Cas*]i mobilization in monocytes, macrophages and platelets (4, 14). Changes in intracellular [Ca2+1 induced by 1OnM PAF were measured during VitDs-induced differentiation between day 0 and day 7 (Fig. 1). No substantial response in terms of [Caz+]i variations occurred during the first 4 days of the differentiation process. Day 4.5 represented a break point, since a transient rise of 200nM in [Cag+]i was observed upon addition of PAF. The cell response to PAF increased gradually as a function of increasing incubation time. Pre-exposure of the cells at day 7 to the PAF-specific antagonist WEB 2086 selectively abolished the PAF-mediated response. In contrast, FMLP was still able to induce a transient rise in [Cag+]i in the case of WEB 2086-pretreated HL-60 cells. This result indicates that the observed effects on [Caz+]i mobilization upon PAF stimulation are specific and mediated through a PAF-specific receptor. PAF-R mRNA expression in HI&O cells. We analyzed the expression of the human PAF receptor gene in undifferentiated and differentiated (day 7) HL-60 cells. To this aim, we isolated the PolyA+ fraction of total RNA from VitDa-stimulated and unstimulated cells. The Northern blot of the mRNA isolated from these two populations was hybridized under low stringency conditions with the complete open reading frame of the heterologous cDNA probe of guinea pig PAF-R. Figure 2 shows that undifferentiated HL-60 cells (day 0) did not express detectable levels of corresponding PAF-R mRNA. In contrast, HL-60 cells, exposed to VitD3 for 7 days, expressed a mRNA transcript of approximately 4 kb, clearly crosshybridizing with the guinea pig PAF-R cDNA. These results indicate that a PAF-R gene, with homology to that found in several guinea pig tissues, is expressed in HL-60 cells differentiated into macrophages. Furthermore, PAF-R gene expression is induced during VitDs-driven HL-60 differentiation. To determine the time point of onset of the PAF receptor gene expression, we analyzed the presence of PAF-R specific transcripts at day 1.5 and day 4 of VitDsdriven differentiation. Cell cultures were the same as those used for the [Ca2+]i mobilization studies described above. Northern blot analysis of the PolyA+ 1582

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700 -

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m. Effects of 10&I PAF on [Ca2+l; mobilization at different stagesof HL-60 cell differentiation. Five million cells, loaded with Fura2-AM during 1 hour, were washed 5 times with HBSS and supplemented with 1.5 mM CaC12 10 minutes prior to stimulation. The figure illustrates single but representative events out of >lO performed. Treatment of day 7 cells with the PAF antagonist WEB 2086 (10-s M) abolished PAF-induced [Caz+]i mobilization but did not prevent the FMLPinduced response. 1583

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lu-60 PAF-R-

day:

2&sPAF-R-

18s -

GAPDH

4

w

288-

-

GAPDH -

-r-e

2, Northern blot analysis of PAF-R expression in HL-60 cells. Human

rRNA was used as a size marker.

Lower panels show hybridization

to GAPDH

probe, indicating equivalent loading and integrity of the RNA. A. Ten ug of PolyA+ RNA from undifferentiated (day 0) and VitDg-differentiated HL-60 cells (day 7) were hybridized with a heterologous guinea pig PAF-R probe of 895 bp covering

the almost entire open reading frame (8). A main band at approximately

4 kb

indicates the presence of a corresponding PAF-R transcript in differentiated, but not in undifferentiated, HL-60 cells. B. PolyA+ RNA isolated from day 1.5 and day

4-differentiated HL-60 cells was analyzed as described above. As hybridization probe we used the complete guinea pig PAP-R cDNA of 3.026 kb.

fractions hybridized with the entire guinea pig PAF-R cDNA showed the presence of a weak 4 kb transcript in day 4differentiated HL-60 cells. No transcript was seen at day 1.5. These results parallel those of PAF-dependent [Ca2+li mobilization. They further suggest that the PAF-R gene expression is induced between day 1.5 and day 4 of differentiation and that this is followed by functional expression of PAF-R on the cell surface. PAF’-R expmssion in TRP-1 cells. To test whether VitDs may be directly involved in the modulation of PAF-R gene expression, we used the monocytic cell line THP-1, which constitutively expresses PAF-R on its surface (15). We cultured the cells in the presence of VitD3 for 4 days, a period corresponding to that needed for initial evidence of PAF-R gene expression in HL-60 cells. The response in terms of [Ca2+]i variation upon stimulation with 1OnM PAF, which was selectively inhibited by WEB 2086, confirmed the presence of functional and specific PAF receptors on the surface of THP-1 cells (Fig. 3A). Northern blot analysis of PolyA+ fractions of RNA from stimulated and unstimulated THP-1 cell cultures showed that the 4 kb PAP-R transcript was expressed at a very low, but constant level (Fig. 3B). This result indicates that VitD3 is not able to directly enhance transcription of this PAF receptor in THP-1 cell line. 1584

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THP-

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fipure 3, A. [Caz+]i mobilization in THP-1 cells exposed to 10 nM PAF. Conditions were as described in Figure 1. B. Northern analysis of 16 clg of PolyA+ RNA from VitDa-stimulated (+I or unstimulated (-1 THP-1 cells show very low but constant levels of PAF-R transcripts with a length of approximately 4 kb. The PAF-R probe was the same as in Figure 2B. Human rRNA was used as a size marker. Lower panel shows hybridization to GAPDH probe, indicating equivalent loading and integrity of the RNA.

DISCUSSION Data presented here provide the first molecular evidence for modulation of PAF-R expression during the VitDS-dependent differentiation of HL-60 cells towards the macrophage phenotype. Northern blot analysis revealed that PAF-R gene transcription in differentiating HL-60 cells occured from day 4 onward. Prior to this incubation time, HL-60 cells failed to respond to any concentration of PAP, in terms of either [Ca2+]i flux or TNF’ct production (4). Soon after (day 4.51, however, the onset of the [Ca2+]; response to 10 nM PAF was observed. The amount of the transcripts as well as the response in terms of [Cas+]i mediated by 10 nM of PAP increased then gradually toward the end of monocytic differentiation (day 7). The induction of the PAF-R gene transcription preceded the presence of functional PAF receptors at the cell surface as indicated by the increase in [Ca2+]i fluxes in response to PAF. For this reason the PAF-R gene expression must be regulated, at least partially, at the transcriptional level. We showed for THP-1 cells, which constitutively express PAF-R mRNA, that VitD3 per se did not affect transcription of this gene. Therefore, differentiation factors other than VitD3 may interact with the receptor gene and induce expression. Investigations to assess this most interesting aspect of gene regulation are under way. Similar modulation of the 1585

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expression of PAF receptors has been reported in DMSO-induced granulocyte differentiation of HL-60 cells (10). In this system, the receptors are present at the cell surface upon day 5 (9). In contrast to the late expression of PAF-R, the first maturational changes in HL60 cells upon VitDs-induced differentiation occur already between 4 and 24 hours upon exposure, like e.g.: expression of cellular oncogenes (16, for review), lysozyme synthesis, secretion of a-naphthylacetate esterase activity and appearance of monocyte-associated cell surface antigens (e.g. 63D3 and Mac-120, 17). Some of these factors may be implicated in myeloid cell differentiation and have therefore to be expressed at the beginning of the maturation process. Since the expression of PAF-R occurs later on, the receptor may rather represent a marker of mature monocyte, macrophages or granulocytes and its induction a consequence of cell maturation and differentiation. In conclusion, our studies provide an interesting system for studying the PAF-R gene expression as a function of monocyte/macrophage differentiation and may allow better understanding of the physiology of developing cells. AcknowledPments, This work was supported by a grant from the Medical Research Council of Canada and by a post-doctoral fellowship (E.M.) supported by the National Swiss Foundation. The authors thank Dr. C. Dubois for critical reading of the manuscript and D. Bolduc for excellent technical assistance.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. ii14: 15. 16. 17.

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