Macrophage colony stimulating factor regulation by nuclear factor kappa B: a relevant pathway in human immunodeficiency virus type 1 infected macrophages

Abstract

Macrophage colony stimulating factor (M-CSF) is a cytokine that promotes monocyte differentiation and survival. When overexpressed, M-CSF contributes to pathology in a wide variety of diseases, including osteoporosis, obesity, certain human cancers, and in human immunodeficiency virus type 1 (HIV-1) infection, particularly with respect to monocyte/macrophage infection and the development of HIV-1 associated central nervous system disorders. In this study, our aim was to expand the current knowledge of M-CSF regulation, focusing on nuclear factor kappa B (NF-κB), a transcription factor playing a prominent role during inflammation and HIV-1 infection. Our results suggest that tumor necrosis factor alpha (TNF-α) promotes M-CSF secretion in primary macrophages and activates the -1310/+48 bp M-CSF promoter in Mono-Mac 1 cells. Inhibitors of the NF-κB pathway diminish this response. We identified four putative NF-κB and four CCAAT-enhancer-binding protein beta binding sites within the M-CSF promoter. Our findings, using promoter constructs mutated at individual NF-κB sites within the M-CSF promoter region, suggest that these sites are redundant with respect to NF-κB regulation. TNF-α treatment promoted NF-κB p65 binding to the M-CSF promoter in phorbol 12-myristate 13-acetate (PMA) treated U937 cells chronically infected with HIV-1 (U1 cells), but not in PMA treated uninfected U937 cells, suggesting that the presence of HIV-1 increases the NF-κB response. In conclusion, our findings demonstrate that NF-κB induces M-CSF expression on a promoter level via multiple functional NF-κB binding sites and that this pathway is likely relevant in HIV-1 infection of macrophages.

FIG. 1.

The M-CSF promoter contains four C/EBP and NF-κB sites. The M-CSF promoter (−1310/+48 bp) was amplified from human genomic DNA and ligated into the pGL3-basic vector (Promega Corporation) to form plasmid pGL3-M-CSF. Mutations in the putative C/EBPβ and NF-κB binding sites in the M-CSF promoter were generated by amplification of the whole plasmid pGL3-M-CSF using site-directed mutated primers. M-CSF, macrophage colony stimulating factor; NF-κB, nuclear factor kappa B; C/EBPβ, CCAAT-enhancer-binding protein beta.

FIG. 2.

M-CSF production in macrophages is NF-κB responsive. (A) Macrophages were cultured for 3 days of with dexamethasone (Dex) (0.1 μM), RU486 (1 μM), or both treatments. M-CSF production shown, standardized by using protein content. (B) Three days macrophage culture in a TNF-α (10 ng/mL) context in combination with Dex (0.1 μM), RU486 (1 μM), and SC-514 (50 μM) treatments. (C) Dose curve for Dex (0–0.001 μM) on macrophage M-CSF production with and without TNF-α (10 ng/mL). (D) Western blot analysis of IκBα phosphorylation and degradation shown in response to treatment of macrophages with Dex, TNF-α, or combined treatment for 10 min. [M: Media; T: TNF-α (10 ng/mL); D; Dex (0.1 μM); R: RU486 (1 μM)]. (E) Quantification of values from D standardized by levels of β-actin. (Student's t-test: *p≤0.05, **p≤0.001, ns: nonsignificant). TNF, tumor necrosis factor.

FIG. 3.

M-CSF is NF-κB responsive in Mono-Mac 1 cells. (A) Effect of 3 days of TNF-α (10 ng/mL) on levels of M-CSF excretion in Mono-Mac 1 cells. (B) Western blot analysis of NF-κB p65 nuclear translocation after 1 h of treatment with media alone, Dex (0.1 μM), RU486 (1 μM), or TNF-α (10 ng/mL). Cytoplasmic (C) and Nuclear (N) fractions are shown. Lamin A is used as a positive control for nuclear extract. (C) Quantification of B with cytoplasmic fractions standardized for β-actin and the nuclear fractions standardized by Lamin A levels. The standardized nuclear and cytoplasmic values were then divided to determine the ratio of nuclear/cytoplasmic levels of NF-κB p65 in each condition.

FIG. 4.

The M-CSF promoter contains functional redundant NF-κB binding sites. (A) M-CSF promoter response of single NF-κB mutants (NF-κB M1-M4) to 1 day of TNF-α treatment in Mono-Mac 1 cells as determined by luciferase activity. Firefly luciferase values were standardized for transfection by using Renilla luciferase values from the same sample. M-CSF promoter response of quadruple NF-κB, C/EBPβ, or both promoter element mutants (NF-κB D4, C/EBP D4, and pMCSF D8, respectively) to 1 day of TNF-α (B) or Dex (C) treatment in Mono-Mac 1 cells. (Student's t-test: *p≤0.05, **p≤0.001, ns: nonsignificant).

FIG. 5.

NF-κB p65 binds to the M-CSF promoter in myeloid cell lines. (A) ChIP analysis of NF-κB p65 binding to the M-CSF promoter in undifferentiated and PMA (5 nM) differentiated U937 in response to 1.5 h of TNF-α treatment. Amplification shown at 45 cycles of PCR. B. NF-κB ChIP for M-CSF promoter in chronically infected, PMA differentiated U1 cells in response to TNF-α (B). Amplification shown at 38 cycles. (C) NF-κB ChIP for M-CSF promoter in Mono-Mac 1. Amplification shown at 45 cycles. (D) Levels of M-CSF expression than U937 cells after 1 day of PMA treatment followed by 3 days of TNF-α treatment (10 ng/mL) (D). (Student's t-test: *p≤0.05, **p≤0.001, ns: nonsignificant). PMA, phorbol 12-myristate 13-acetate; ChIP, chromatin immunoprecipitation.