Multiple Ets Factors and Interferon Regulatory Factor-4 Modulate CD68 Expression in a Cell Type-specific Manner

Abstract

CD68 is a transmembrane glycoprotein expressed in all cells of the mononuclear phagocyte lineage including monocytes and tissue resident macrophages. Deletion analysis of the 5-flanking sequences of the gene demonstrated that the proximal ؊150-bp sequence of the CD68 promoter exhibits high level promoter activity in macrophages. Mutations that abolish Ets factor binding at positions ؊106 and ؊89 reduce promoter activity in macrophages to 12 and 30%, respectively. Band shift experiments show that PU.1 associates with the ؊89 site whereas, Elf-1 preferentially binds the ؊106 Ets binding site and enhances CD68 activity in vitro. Furthermore, chromatin immunoprecipitation experiments confirm that Elf-1 and PU.1 associate with the CD68 proximal promoter in vivo in THP-1 cells. PU.1 does not bind to the CD68 promoter alone but instead forms heterocomplexes with members of the interferon regulatory factor family (IRF) including IRF-4 and IRF-8. IRF-4 and IRF-8 typically mediate transcriptional activation when associated with PU.1 on composite elements. However, our data show that PU.1/IRF-4 and IRF-8 heterocomplexes down-regulate CD68 promoter activity in macrophages and repression is dependent on the integrity of both the IRF and PU.1 half-sites of this composite element. Chromatin immunoprecipitation data reveal that neither IRF-4 nor IRF-8 associate with the CD68 proximal promoter in macrophages in vivo but IRF-4 is associated with the promoter in B lymphocytes. We propose that expression of CD68 in myeloid cells requires the Ets transcription factors Elf-1 and PU.1 and CD68 expression is down-regulated in lymphoid cells by combinatorial interactions between PU.1 and IRF-4.

PU.1 Regulates the Tissue-specific Expression of Dendritic Cell-specific Intercellular Adhesion Molecule (ICAM)-3-grabbing Nonintegrin

“…Immunoprecipitating antibodies included affinity-purified rabbit polyclonal antibody against human PU.1 (sc-352; Santa Cruz Biotechnology) and purified polyclonal rabbit IgG as a control. As a positive control, amplification of the CD68 promoter was achieved with oligoncleotides 5Ј-CAACTGCCCTAG-GACTCCGTTTG-3Ј and 5Ј-TCAGCCTCCTTCCTCCTTACCT-3Ј, which together amplify a 318-bp region (33).…”

CD68/macrosialin: not just a histochemical marker

“…E26 transformation-specific related transcription factor (Elf-1) and Friend leukemia integration 1 transcription factor (Fli-1), also cooperates with PU-1 in priming CD68 expression. 23 The proximal -150-bp sequence of the mouse CD68 promoter exhibits high level promoter activity in macrophages. Ets factors (Elf-1 and Fli-1) associate with the -106-bp site while PU.1 binds to the -89-bp site that represents a composite element also recognized by interferon-regulatory factors (IRFs).…”

“…Ets factors (Elf-1 and Fli-1) associate with the -106-bp site while PU.1 binds to the -89-bp site that represents a composite element also recognized by interferon-regulatory factors (IRFs). 23 In hematopoietic cells, IRF-4 and IRF-8 are specifically expressed driving development of myeloid and lymphoid lineages. Both IRFs are able to form heterocomplexes with PU.1.…”

“…Chromatin immunoprecipitation data revealed that neither IRF-4 nor IRF-8 associate with the CD68 -89 site in macrophages in vivo but IRF-4 is associated with this site in B cells causing inhibition of CD68 expression. 23 In addition to high expression in the mononuclear phagocyte lineage, considerably lower levels of CD68 are expressed in lymphoid cells such as CD19 + B lymphocytes and CD4 + T lymphocytes. CD68 expression was also found in granulocytes such as basophils (taken from patients with myeloproliferative disorders) and intestinal neutrophils derived from inflamed sites of patients with inflammatory bowel disease.…”

“…45 CD68 was also considered as a member of the family of SRs as a scavenger receptor type D (SCARD) 46 because (i) CD68 can be significantly upregulated in macrophages responding to inflammatory stimuli; 23,36 (ii) Figure 1 A scheme shows the general features of the domain organization of murine macrosialin and human proteins LAMP-1, LAMP-2, DC-LAMP, and CD68 and depicts the principal differences between the family members. N-glycans and O-glycans are indicated by red and green Ys, respectively.…”