Induction of lactoferrin gene expression in myeloid or mammary gland cells by human T-cell leukemia virus type 1 (HTLV-1) tax: implications for milk-borne transmission of HTLV-1

Abstract

Human T-cell leukemia virus type 1 (HTLV-1), the causative agent of adult T-cell leukemia, is transmitted vertically via breastfeeding. We have previously demonstrated that lactoferrin, a major milk protein, enhances HTLV-1 replication, at least in part by upregulating the HTLV-1 long terminal repeat promoter. We now report that HTLV-1 infection can induce lactoferrin gene expression. Coculture with HTLV-1-infected MT-2 cells increased the levels of lactoferrin mRNA in myeloid-differentiated HL-60 cells, as well as MCF-7 cells, models of two probable sources (neutrophils and mammary epithelium) of lactoferrin in breast milk. MT-2 cell coculture could be replaced with cell-free culture supernatants of MT-2 cells to exert the same effect. Furthermore, extracellularly administered Tax protein also induced lactoferrin gene expression at physiologically relevant concentrations. In transient-expression assays, Tax transactivated the lactoferrin gene promoter in HL-60 or MCF-7 cells. Experiments with Tax mutants, as well as site-directed mutants of the lactoferrin promoter reporters, indicated that the NF-kappaB transactivation pathway is critical for Tax induction of the lactoferrin gene promoter activity in myeloid-differentiated HL-60 cells, but not in MCF-7 cells. These results suggest that HTLV-1 infection may be able to induce expression of lactoferrin in a paracrine manner in the lactic compartment. Our findings, in conjunction with our previous study, implicate that mutual interaction between HTLV-1 and lactoferrin would benefit milk-borne transmission of this virus.

FIG. 1.

HTLV-1 Tax induces lactoferrin gene expression. (A) MT-2 cell coculture or MT-2 cell-free culture supernatants upregulated lactoferrin gene expression in DMSO-differentiated HL-60 cells or MCF-7 cells. Total RNA was extracted from untreated cells (open bars) or cells that had been cocultured with MT-2 cells through a semipermeable membrane filter (closed bars) or incubated in 50% MT-2 cell-free supernatants (hatched bars) for 2 days and was subjected to real-time RT-PCR of lactoferrin or tubulin gene mRNA. Lactoferrin mRNA expression relative to that of tubulin mRNA in untreated cells is shown as 1. The results are means ± standard deviations (SD) from three independent experiments. (B) Recombinant Tax protein upregulated lactoferrin gene expression. Expression of lactoferrin mRNA relative to that of tubulin mRNA was calculated for total RNA extracted from DMSO-differentiated HL-60 cells or MCF-7 cells that had been treated with GST (1.5 nM) (open bars), GST (1.2 nM) plus GST-Tax (0.3 nM) (closed bars), or GST-Tax (1.5 nM) (hatched bars) for 16 h, as in panel A. (C) Chloroform extraction abolished the GST-Tax effect on lactoferrin gene expression. DMSO-differentiated HL-60 cells were treated with GST (1.5 nM) (open bar), control nonextracted GST-Tax (1.5 nM) (closed bar), or chloroform-extracted GST-Tax (1.5 nM) (hatched bar). The results are means ± SD from triplicate experiments. (D) Neutralization of Tax with anti-Tax serum markedly reduced the ability of MT-2 cell-free culture supernatant or GST-Tax preparations to induce lactoferrin gene expression. (Left) DMSO-differentiated HL-60 cells were incubated with Jurkat cell-free culture supernatant (open bar) or with MT-2 cell-free culture supernatants plus normal rabbit serum (closed bar) or anti-Tax serum (hatched bar). (Right) Similarly, DMSO-differentiated HL-60 cells were treated with GST (1.5 nM) (open bar), GST-Tax (1.5 nM) plus normal rabbit serum (closed bar), or GST-Tax (1.5 nM) plus anti-Tax serum (hatched bar). The results are means ± SD from triplicate experiments. (E) Tax-inducible soluble factors played a role in inducing lactoferrin gene expression. DMSO-differentiated HL-60 cells were treated with GST or GST-Tax for 2 h and washed extensively to remove it, and cell-free culture supernatants were collected after 24 h of incubation. Another line of DMSO-differentiated HL-60 cells were exposed to cell-free culture supernatants from GST (open bar)- or GST-Tax (closed bar)-preconditioned HL-60 cells for 16 h, and total RNA was extracted from those cells for real-time RT-PCR analysis. The results are means ± SD from triplicate experiments.

FIG. 2.

Mutual interactions between HTLV-1 and mammary epithelial cells. MCF-7 cells were treated with GST or GST-Tax for 2 h and washed extensively to remove it, and cell-free culture supernatants were collected after 24 h of incubation. HeLa-Luc cells were exposed to cell-free culture supernatants from GST (open bar)- or GST-Tax (closed bar)-preconditioned MCF-7 cells for 24 h, and cell lysates were subjected to luciferase assays. The results are means plus standard deviations from triplicate experiments.

FIG. 3.

HTLV-1 Tax transactivates the lactoferrin promoter. (A) DMSO-treated HL-60 cells or MCF-7 cells were transfected with pGL-Lf; treated with GST (1.5 nM) (open bars), GST (1.2 nM) plus GST-Tax (0.3 nM) (closed bars), or GST-Tax (1.5 nM) (hatched bars) for 2 days; and subjected to luciferase assays. Fold induction is the luciferase activity relative to that obtained with GST treatment. The results are means ± standard deviations from three independent experiments. (B) DMSO-pretreated HL-60 cells were transfected with 20 μg of pGL-Lf, along with pMT-2T (10 μg) (open bar), pMT-2T (8 μg) plus pMT-Tax (2 μg) (closed bar), or pMT-Tax (10 μg) (hatched bar). On the other hand, MCF-7 cells were transfected with 10 μg of pGL-Lf, along with pMT-2T (1 μg) (open bar), pMT-2T (0.8 μg) plus pMT-Tax (0.2 μg) (closed bar), or pMT-Tax (1 μg) (hatched bar). Fold induction is the luciferase activity relative to that obtained with pGL-Lf plus pMT-2T. The results are from four independent experiments.

FIG. 4.

Identification of cis-acting elements on the human lactoferrin gene promoter. (A) Schematic diagram of the human lactoferrin gene promoter. The horizontal arrow indicates the transcription start site (13), and numbers above or below the sequence indicate nucleotides relative to the transcription start site. cis-acting elements demonstrated in this study (NF-κB and AP1) are underlined, and references are indicated for those previously reported. (B and C) Identification of binding sites for NF-κB and AP1. (B) LfNF-κB probe was incubated with nuclear extracts from MCF-7 cells that had been pretreated with phorbol 12-myristate 13-acetate (1 μM) and ionomycin (1 μM) for 15 min. Where indicated above the figure, a 100-fold molar excess of unlabeled oligonucleotides (lanes 3 to 6) (Table 1) or antibodies (lanes 8 to 11) was added to the reaction mixtures. Lane 1 represents probe alone, and lanes 2 to 11 represent reactions in the presence of nuclear extracts. FP indicates free probe. Open and solid arrows indicate the p65/p50 heterodimer (NF-κB) and p50 homodimer, respectively. The triangle indicates supershifted p50-containing complex. The open circle indicates nonspecific complex, and the closed circle indicates another, uncharacterized DNA-protein complex, which may be related to Rel family protein but apparently contains neither p50, p65, nor c-Rel. Similar results were obtained with DMSO-pretreated HL-60 cell nuclear extracts. (C) Lf-AP1-1 probe was incubated with MCF-7 nuclear extracts, as in panel B. Where indicated above the figure, a 100-fold molar excess of nonlabeled oligonucleotides (lanes 3 to 5) (Table 1) or antibodies (lanes 7 and 8) was added to the reaction mixtures. Lane 1 represents probe alone, and lanes 2 to 8 represent reactions in the presence of nuclear extracts. Solid arrows and open circles indicate Fos/Jun heterodimer (AP-1) and nonspecific complexes, respectively. Similar results were obtained with Lf-AP1-2 probe.

FIG. 5.

Cell-type-dependent regulation of the lactoferrin promoter activity by Tax. (A) Transactivation of HIV-1 LTR or HTLV-1 LTR by HTLV-1 Tax or its mutants. Five million PBMC from healthy volunteers were transfected with pU3R-luc (1 μg) or pGL-HIV-1-LTR (2 μg), along with 1 μg of pcDNA2.1, pCG-Tax, pCG-M22, or pCG-Δ3. Luciferase activity was measured 2 days after transfection. Fold induction is the luciferase activity relative to that obtained with pcDNA2.1. The results are means ± standard deviations (SD) from three independent experiments. (B) Transactivation of the human lactoferrin promoter by HTLV-1 Tax or its mutants. DMSO-pretreated HL-60 cells or MCF-7 cells were transfected with pGL-Lf, along with pcDNA2.1, pCG-Tax, pCG-M22, pCG-Δ3, or a combination of the two mutants (M22 + Δ3). Fold induction is the luciferase activity relative to that obtained with pcDNA2.1. The results are means ± SD from three independent experiments. (C) Tax activation of the lactoferrin promoter site-directed mutants. DMSO-pretreated HL-60 cells or MCF-7 cells were transfected with pGL-Lf or its derivatives, along with pMT-2T or pMT-Tax. Fold induction is the luciferase activity relative to that obtained with pGL-Lf plus pMT-2T. The results are means ± SD from three independent experiments. (D) Tax induction of NF-κB binding activity in myeloid-differentiated HL-60 cells or MCF-7 cells. DMSO-pretreated HL-60 cells (top) or MCF-7 cells (bottom) were incubated with GST or GST-Tax for 30 min, and nuclear extracts were prepared for gel mobility shift assays using Lf-NF-κB (left) or Sp1-C (right). Open and solid arrows indicate p65/p50 heterodimer (NF-κB) and p50 homodimer, respectively. The closed circle indicates Sp1 complex.