The Retinoic Acid-responsive Proline-rich Protein Is Identified in Promyeloleukemic HL-60 Cells

Abstract

To identify new genes that retinoic acid activates, we employed an mRNA differential display technique and screened for genes that are differentially expressed in promyeloleukemic HL-60 cells incubated in the presence of all-trans-retinoic acid (ATRA) compared with the absence of ATRA. We cloned the coding region of a retinoic acid-induced gene from a human thymus library, which was the mRNA encoding the 666-amino acid human homologue of mouse proline-rich protein 76. We have designated it RARP1 (retinoic acid response proline-rich protein 1). Transcription of an ϳ2.4-kbp mRNA occurred mainly in organs with immune functions, such as thymus, spleen, and peripheral leukocytes. Cycloheximide blocked the ATRA-induced expression. In megakaryocyte-like human erythroleukemia HEL cells, the amount of RARP1 mRNA was high, but it was low in human T-lymphoblastoid Jurkat cells. A specific antibody against RARP1 recognized a 110-kDa protein, which accumulates after incubation of HL-60 cells with ATRA. In immunohistochemical experiments, strong RARP1 staining was observed in the megakaryocytes of bone marrow and spleen, and heterogeneous stain was seen in thymus. Transcriptional studies showed that RARP1 expression impaired the transactivation through activator protein1 and serum responseelement in all cell lines we checked, whereas it did not affect the transactivation through cAMP-response element in the same cell lines. Further analysis demonstrated that proline-rich regions of RARP1 are the functional regions regulated for suppression of activator protein1 transactivation. These data suggest that ATRAinducible RARP1 selectively affects signal transduction and may contribute to myeloid and megakaryocytic differentiation.Retinoids and their active metabolites, including all-transretinoic acid (ATRA), 1 play an important role in morphogenesis, differentiation, and homeostasis (1). In the hematopoietic system, Breitman et al. (2) first reported the action of ATRA to promote HL-60 cell differentiation in vivo. The specific action has led to widespread use of ATRA to promote induction of neutrophilic differentiation of leukemic cells as a selective means of chemotherapy for fresh acute promyeloleukemic cells (3). Retinoic acid receptors and retinoic X-receptors are two distinct families of nuclear receptors that belong to the steroid/ thyroid hormone receptor family (4). ATRA binds with retinoic acid receptors, and another retinoid, 9-cis-retinoic acid, binds with retinoic X-receptors (5). DNA array, suppression-subtractive hybridization, and differential display-PCR analyses demonstrated that some genes are up-and down-regulated directly or indirectly by the treatment with ATRA in human promyeloleukemia NB4 cells (6). Thus, it is understandable that retinoic acid directly or indirectly activates a series of genes that may contribute to differentiation of HL-60 cells.To identify the gene that responds to retinoic acid, we employed a differential display technique using the mRNA from HL-60 cells treated with or without ATRA. We...

RIAM (Rap1-interacting adaptor molecule) regulates complement-dependent phagocytosis

“…In this cell line, RARP-1 was proposed to play a role in myeloid differentiation based on its capacity to regulate transcriptional activity [27]. The HL-60 cell line has been used in opsonophagocytic assays using bacteria or RBCs as phagocytic particles [28], hence we decided to use this cell line as a model to study the role of RIAM in complement-mediated phagocytosis.…”

“…This finding was observed also in the shRIAM-99 cell line albeit to a different magnitude (Supplemental Figure S1a). RARP1/RIAM was proposed to play a role in differentiation [27]. Because expression of CR3 and CR4 receptors is upregulated during myeloid differentiation [29, 30], first, we examined whether RIAM might have a role in the induction of these phagocytic receptors.…”

The adaptor molecule RIAM integrates signaling events critical for integrin-mediated control of immune function and cancer progression

“…This interaction is mediated by the WW (tryptophan-tryptophan) domain of Fe65 interacting with the proline-rich regions of RIAM (2). In an independent study, the gene encoding RIAM was also identified as transcriptionally induced in response to all-trans retinoic acid (ATRA) in the promyeloleukemic HL-60 cell line, and the protein was accordingly named retinoic acid-responsive proline-rich protein 1 (RARP-1) (3). In that system, it was found that forced expression in various cell types suppressed transactivation of activator protein 1 (AP-1) and serum response element (SRE), leading to the conclusion that this protein was functionally involved in cell growth arrest.…”

“…Nonhematopoietic tissues, in which the RIAM transcript has been identified, include heart, brain, lung, liver, skeletal muscle, kidney, and pancreas. The abundance of the RIAM protein varies among tissues and cell types, but the greatest amount is detected in cells of hematopoietic origin (1,3,4).…”

Copy Number Deletion Has Little Impact on Gene Expression Levels in Racehorses

“…DIAPH3 and PAK7 genes (Supplementary Figure 1a) are involved in the ‘regulation of actin cytoskeleton’ term of the KEGG pathway. The ANK1 gene encodes Ankyrins, which are a family of proteins that link membrane proteins to the cytoskeleton (Chorzalska et al, 2010), and the APBB1IP gene functions in signal transduction from Ras activation to cytoskeleton remodeling (Inagaki et al, 2003). These cellular-structure-related genes were not likely under tight transcriptional regulation, which could be due to the buffering effect of those genes on dosage alteration.…”