Down-Regulation of P_2U-Purinergic Nucleotide Receptor Messenger RNA Expression DuringIn VitroDifferentiation of Human Myeloid Leukocytes by Phorbol Esters or Inflammatory Activators

Abstract

HL-60 human promyelocytic leukocytes express G protein-coupled P2U-purinergic nucleotide receptors (P2UR or P2Y2R) that activate inositol phospholipid hydrolysis and Ca24 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 approximately 60 min, and this was not affected by acute exposure (< or = 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 gamma-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.

International Union of Pharmacology LVIII: Update on the P2Y G Protein-Coupled Nucleotide Receptors: From Molecular Mechanisms and Pathophysiology to Therapy

“…HeLa cell proliferation in response to P2Y 2 receptor activation is associated with the PI3-K-and ERK1/2-dependent expression of the early response protein c-fos (Muscella et al, 2003). Consistent with a role for P2Y 2 receptors in cell proliferation, P2Y 2 receptor mRNA expression is down-regulated during cell differentiation (Martin et al, 1997). The P2Y 2 receptor is up-regulated in thymocytes as an immediate early gene response (Koshiba et al, 1997) and in injured and stressed tissues Seye et al, 2002), and nucleotides induce ERKdependent astrocyte proliferation under a variety of conditions including stretch-or stab-induced injury (Neary et al, 1994(Neary et al, , 1996(Neary et al, , 1999Franke et al, 1999).…”

Regulation of delta opioid receptor expression by anti-CD3-ε, PMA, and ionomycin in murine splenocytes and T cells

“…Nuclear extracts from PMA-treated cells showed increased binding to the TRE in the transferrin receptor gene promoter region that was abolished by an oligonucleotide containing the consensus TRE sequence. Another study determined the effect of PMA on the expression of mRNA encoding the seven-transmembrane G-protein-coupled P2U-purinergic nucleotide receptor in HL-60 cells [31]. P2UR mRNA levels declined 5-to 10-fold with PMA and their half-life was unaffected.…”

Molecular mechanism of nucleotide-induced primary granule release in human neutrophils: role for the P2Y2receptor

“…However, subsequent work in the P2 receptor field has identified other P2Y receptors that can be stimulated by ATP and/or UTP (eg., P2Y 4 , P2Y 6 , P2Y 11 ) (37). Molecular characterization of P2Y receptor subtypes in human neutrophils and differentiated HL-60 cell line by RT-PCR analysis revealed that granulocytic cells express P2Y 2 , P2Y 4 , P2Y 6 , and P2Y 11 but not the P2Y 1 receptor (9,13,14,24,27,33,50). We undertook the characterization of the P2Y receptor subtypes in human neutrophils by functional approaches by using intracellular calcium increases as an early signaling event downstream of P2Y receptor activation.…”

“…Except for the P2Y 1 receptor, there are no specific antagonists for the P2Y receptors. RT-PCR analysis revealed that human neutrophils express P2Y 2 , P2Y 4 , P2Y 6 , and P2Y 11 receptors (9,13,14,24,27,33,50). P2Y 1 , P2Y 4 , and P2Y 6 couple to G q to activate phospholipase C, causing formation of inositol-1,4,5-triphosphate and mobilization of intracellular Ca 2ϩ (15,18).…”