Down-Regulation of P2U-Purinergic Nucleotide Receptor Messenger RNA Expression During In Vitro Differentiation of Human Myeloid Leukocytes by Phorbol Esters or Inflammatory Activators
- Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4970
Abstract
HL-60 human promyelocytic leukocytes express G protein-coupled P2U-purinergic nucleotide receptors (P2UR or P2Y2R) that activate inositol phospholipid hydrolysis and Ca2+ mobilization in response to ATP or UTP. We examined the expression of functional P2UR and P2UR mRNA levels during in vitro differentiation of HL-60 cells by dibutyryl-cAMP (Bt2cAMP), which induces a granulocyte/neutrophil phenotype, or by phorbol-12-myristate-13-acetate (PMA), which induces a monocyte/macrophage phenotype. Both P2UR function and P2UR mRNA levels were only modestly attenuated during granulocytic differentiation by Bt2cAMP. In contrast, P2UR function, as assayed by either Ca2+ mobilization or inositol trisphosphate generation, was greatly reduced in PMA-differentiated cells. This inhibition of P2UR function was strongly correlated with PMA-induced decreases in P2UR mRNA levels, as assayed by Northern blot analysis or reverse transcription-polymerase chain reaction-based quantification. Although PMA induced an early, transient up-regulation of P2UR mRNA, this was rapidly followed by a sustained decrease in P2UR mRNA to a level 5–10-fold lower than that in undifferentiated HL-60 cells. The half-life of the P2UR transcript in HL-60 cells was ∼60 min, and this was not affected by acute exposure (≤4 hr) to Bt2cAMP or PMA. PMA down-regulated P2UR mRNA in THP-1 monocytes and HL-60 granulocytes but not in A431 human epithelial cells or human keratinocytes. P2UR mRNA was also down-regulated in THP-1 monocytes differentiated into inflammatory macrophages by γ-interferon and endotoxin. These data indicate that myeloid leukocytes possess tissue-specific mechanisms for the rapid modulation of P2UR expression and function during differentiation and inflammatory activation.
Footnotes
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Send reprint requests to: George R. Dubyak, Ph.D., Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4970. E-mail: gxd3{at}po.cwru.edu
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This work was supported by National Institutes of Health Grant GM36387 (G.P.D.). K.A.M. was supported by National Institutes of Health Training Grant HL07678.
- Abbreviations:
- P2UR
- P2Ureceptor(s)
- Bt2cAMP
- dibutyryl-cAMP
- PMA
- phorbol-12-myristate-13-acetate
- FPR
- formyl peptide receptor
- IL
- interleukin
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
- LPS
- lipopolysaccharide
- PLC
- phospholipase C
- InsP2
- inositol bisphosphate
- GTPγS
- guanosine-5′-O-(3-thio)triphosphate
- InsP3
- inositol trisphosphate
- BSA
- bovine serum albumin
- IFN
- interferon
- RT
- reverse-transcription (or -transcriptase)
- PCR
- polymerase chain reaction
- PK
- protein kinase
- EGTA
- ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- PI
- phosphatidylinositol
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- Received May 29, 1996.
- Accepted September 27, 1996.
- The American Society for Pharmacology and Experimental Therapeutics
