Soluble CD26/Dipeptidyl Peptidase IV Enhances the Transcription of IL-6 and TNF-α in THP-1 Cells and Monocytes

Abstract

CD26 is a 110-kDa multifunctional molecule having dipeptidyl peptidase IV (DPPIV) enzyme activity and is present on the surface of human T cells. Soluble CD26 (sCD26) exists in human blood and enhances the proliferation of peripheral T lymphocytes induced by tetanus toxoid (TT). The mechanisms by which CD26 enhances the activation of T cells and monocytes remain to be fully elucidated. In this study, we compared the stimulation of THP-1 cells and isolated human monocytes with a combination of recombinant sCD26 and lipopolysaccharide (LPS) and the stimulation of these cells with LPS alone. We found that addition of sCD26 increased TNF-α and IL-6 mRNA and protein expression and enhanced ERK1/2 levels in the cytosol as well as c-Fos, NF-κB p50, NF-κB p65, and CUX1 levels in the nuclei of these cells. On the other hand, the selective DPPIV inhibitor sitagliptin inhibited the increase in TNF-α mRNA and protein expression as well as the increase in ERK, c-Fos, NF-κB p50, NF-κB p65, and CUX1 levels. However, it did not inhibit the increase in IL-6 mRNA and protein expression. We then demonstrated that sCD26 enhanced binding of transcription factors to the TNF- and IL-6 promoters and used reporter assays to demonstrate that transcription factor binding enhanced promoter activity. Once again, we observed differential activities at the TNF- and IL-6 promoters. Finally, we demonstrated that CUX-1 overexpression enhanced TNF- production on sCD26/LPS stimulation, while CUX-1 depletion had no effect. Neither CUX-1 overexpression nor CUX-1 depletion had an effect on IL-6 stimulation. These results are discussed in the context of a model that describes the mechanisms by which stimulation of monocytic cells by sCD26 and LPS leads to elevation of TNF- and IL-6 expression. CUX-1 is identified as a new transcription factor that differently regulates the activities of the TNF- and IL-6 promoters.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Effects of LPS and sCD26/DPPIV(+) or sCD26/DPPIV(−) stimulation of THP-1 cells and monocytes on TNF-α and IL-6 mRNA expression, assessed by quantitative RT-PCR.

A, B. Effects of stimulating THP-1 cells with a combination of LPS and sCD26/DPPIV(+) or sCD26/DPPIV(−) on TNF-α and IL-6 mRNA expression. THP-1 cells (1×105) were incubated in the culture medium with or without LPS or various concentrations of sCD26/DPPIV(+) and sCD26/DPPIV(−). The cells were then cultured for the indicated periods. Total RNA was prepared and subjected to quantitative RT-PCR analysis. Each panel shows a typical example of the results. Values represent mean ± S.E.M., n = 3, *P<0.05 vs. control (stimulation with LPS alone). **P<0.05 vs. Mock. C, D. Effects of stimulating monocytes with a combination of LPS and sCD26/DPPIV(+) or sCD26/DPPIV(−) on TNF-α and IL-6 mRNA expression. Monocytes (1×105) were incubated in the culture medium with or without LPS or various concentrations of sCD26/DPPIV(+) or sCD26/DPPIV(−). Then, the cells were cultured for the indicated periods. Total RNA was prepared and subjected to quantitative RT-PCR analysis. Each panel shows a typical example of the results. Values represent mean ± S.E.M., n = 3, *P<0.05 vs. control (stimulation with LPS alone). **P<0.05 vs. Mock. Statistical analysis was performed using Student’s t-test.

Figure 2. Effects of LPS and sCD26/DPPIV(+) or sCD26/DPPIV(−) stimulation of THP-1 cells and monocytes on TNF-α and IL-6 protein secretion, assessed using ELISAs.

A, B. Effects of stimulating THP-1 cells with a combination of LPS and sCD26/DPPIV(+) or sCD26/DPPIV(−) on TNF-α and IL-6 protein secretion. THP-1 cells (1×105) were incubated in the culture medium with or without LPS or various concentrations of sCD26/DPPIV(+) or sCD26/DPPIV(−). The cells were then cultured for the indicated periods. Serum was obtained from the well and subjected to ELISA. Each panel shows a typical example of the results. Values represent mean ± S.E.M., n = 5, *P<0.05 vs. control (stimulation with LPS alone). **P<0.05 vs. Mock. C, D. Effects of stimulating monocytes with a combination of LPS and sCD26/DPPIV(+) or sCD26/DPPIV(−) on TNF-α and IL-6 protein secretion. Monocytes (1×105) were incubated in the culture medium with or without LPS or various concentrations of either sCD26/DPPIV(+) or sCD26/DPPIV(−). The cells were then cultured for the indicated periods. Serum was obtained from the well and subjected to ELISA. Each panel shows a typical example of the results. Values represent the mean ± S.E.M., n = 5, *P<0.05 vs. control (stimulation with LPS alone). **P<0.05 vs. Mock. Statistical analysis was performed using Student’s t-test.

Figure 3. Effects of LPS and sCD26/DPPIV(+) or sCD26/DPPIV(−) stimulation of THP-1 cells and monocytes on ERK1/2, AKT, c-Fos, NF-κB p65, NF-κB p50, and CUX1 protein expression, assessed by Western blotting.

A, B. Effects of stimulating THP-1 cells with a combination of LPS and sCD26/DPPIV(+) or sCD26/DPPIV(−) on ERK1/2, AKT, c-Fos, NF-κB p65, NF-κB p50, and CUX1 protein expression with or without the DPPIV inhibitor sitagliptin. THP-1 cells (1×105) were incubated in culture medium with or without LPS, with or without various concentrations of sCD26/DPPIV(+) or sCD26/DPPIV(−), and with or without the DPPIV inhibitor sitagliptin (1 µM). The cells were then cultured for the indicated periods. Cytoplasmic and nuclear fractions were prepared as described in the Methods section and subjected to Western blotting. Each panel shows a typical example of the results. Values represent mean ± S.E.M., n = 3, *P<0.05 vs. control. C, D. Effects of stimulating monocytes with a combination of LPS and sCD26/DPPIV(+) or sCD26/DPPIV(−) on ERK1/2, AKT, c-Fos, NF-κB p65, NF-κB p50, and CUX1 protein expression with or without the DPPIV inhibitor sitagliptin (1 µM). Monocytes (1×105) were incubated in culture medium with or without LPS, with or without various concentrations of sCD26/DPPIV(+) or sCD26/DPPIV(−), and with or without the DPPIV inhibitor sitagliptin (1 µM). The cells were then cultured for the indicated periods. Cytoplasmic and nucleic fractions were prepared as described in the Methods section and subjected to Western blotting. Each panel shows a typical example of the results. Values represent mean ± S.E.M., n = 3, *P<0.05 vs. control. Statistical analysis was performed using Student’s t-test.

Figure 4. Model for differential regulation of TNF-α and IL-6 gene stimulation by LPS and sCD26/DPPIV(+) or sCD26/DPPIV(−).

Stimulation of cells through TLR4 complexes and sCD26 results in ERK1/2, c-Fos, NF-κB, and CUX1 (arrows). c-Fos, NF-κB, and CUX1 transiently bind to the TNF-α gene promoter but stably bind to the IL-6 gene promoter. sCD26 binds caveolin-1 and is internalized by the MP6/IGF receptor 2. The question mark indicates that the sCD26–caveolin interaction or internalization of sCD26 may stimulate IRAK1 in the TLR4 complex. Further investigations are needed. The question mark also indicates that another DPPIV enzyme-dependent factor or factors may exist to modulate the stability of c-Fos, NF-κB, and CUX1 on the TNF-α and IL-6 gene promoters. B. Diagram showing the structures of human TNF-α (−1.8 kb) and IL-6 (−1.0 kb) reporter constructs. The NF-κB sites are indicated by blue circles, and CUX1 sites are indicated by boxes. The positions of ChIP assay probes and primer pairs are indicated by arrows.

Figure 5. Effects of LPS and sCD26/DPPIV(+) or sCD26/DPPIV(−) stimulation of THP-1 cells and monocytes on the binding affinity of transcription factors with the human TNF-α and IL-6 gene promoters, assessed by ChIP assay.

A, C. Effects of stimulating THP-1 cells with a combination of LPS and sCD26/DPPIV(+) or sCD26/DPPIV(−) on the binding affinity of transcription factors to the human TNF-α and IL-6 gene promoters. THP-1 cells (5×105) were incubated in culture medium with or without LPS, with or without various concentrations of sCD26/DPPIV(+) or sCD26/DPPIV(−), and with or without the DPPIV inhibitor P32/98 (10 µM). The cells were then cultured for the indicated periods. They were fixed and subjected to ChIP analysis. Each panel shows a typical example of the results. Values represent mean ± S.E.M., n = 3, *P<0.05 vs. control. **P<0.05 vs. stimulation by LPS alone. #P<0.05 vs. sCD26/DPPIV(+). B, D. Effects of stimulating monocytes with a combination of LPS and sCD26/DPPIV(+) or sCD26/DPPIV(−) on the binding affinity of transcription factors to the human TNF-α and IL-6 gene promoters. Monocytes (5×105) were incubated in culture medium with or without LPS, with or without various concentrations of sCD26/DPPIV(+) or sCD26/DPPIV(−), and with or without the DPPIV inhibitor P32/98 (10 µM). The cells were then cultured for the indicated periods. They were fixed and subjected to ChIP analysis. Each panel shows a typical example of the results. Values represent mean ± S.E.M., n = 3, *P<0.05 vs. control. **P<0.05 vs. stimulation by LPS alone. #P<0.05 vs. sCD26/DPPIV(+). Statistical analysis was performed using Student’s t-test.

Figure 6. Effects of LPS and sCD26/DPPIV(+) or sCD26/DPPIV(−) stimulation of THP-1 cells and monocytes on the transcriptional activities of the human TNF-α and IL-6 gene promoters.

A, C. Effects of stimulating THP-1 cells and monocytes with a combination of LPS and either sCD26/DPPIV(+) or sCD26/DPPIV(−) on the transcriptional activity of the human TNF-α (−1.8 kb) gene promoter. THP-1 cells (5×105) and monocytes (5×105) were incubated in culture medium with or without LPS or various concentrations of either sCD26/DPPIV(+) or sCD26/DPPIV(−), with or without overexpression of CUX1 or sh-CUX1, and with or without the DPPIV inhibitor sitagliptin (1 µM). The cells were cultured for the indicated periods and then subjected to reporter assays. Each panel shows a typical example of the results. Values represent mean ± S.E.M., n = 3, *P<0.05 vs. control. **P<0.05 vs. stimulation with LPS alone. B, D. Effects of stimulating THP-1 cells and monocytes with a combination of LPS and either sCD26/DPPIV(+) or sCD26/DPPIV(−) on the transcriptional activity of the the human IL-6 gene promoter. THP-1 cells and monocytes (5×105) were incubated in culture medium with or without LPS, with or without various concentrations of either sCD26/DPPIV(+) or sCD26/DPPIV(−), with or without CUX1 or sh-CUX1 overexpression, and with or without the DPPIV inhibitor sitagliptin (1 µM). The cells were cultured for the indicated periods and then subjected to reporter assays. Each panel shows a typical example of the results. Values represent mean ± S.E.M., n = 3, *P<0.05 vs. control. **P<0.05 vs. stimulation with LPS alone. Statistical analysis was performed using Student’s t-test.

Figure 7. Effects of CUX1 and sh-CUX1 overexpression on TNF-α and IL-6 protein expression in THP-1 cells and monocytes stimulated with LPS and sCD26/DPPIV(+) or sCD26/DPPIV(−), assessed by ELISA.

A, C. Effects of CUX1 and sh-CUX1 overexpression on TNF-α protein expression in THP-1 cells and monocytes stimulated with a combination of LPS and sCD26/DPPIV(+) or sCD26/DPPIV(−). THP-1 cells (5×105) or monocytes (5×105) were incubated in culture medium with or without LPS, with or without various concentrations of sCD26/DPPIV(+) or sCD26/DPPIV(−), with or without CUX1 or sh-CUX1 overexpression, and with or without the DPPIV inhibitor sitagliptin (1 µM). The cells were cultured for the indicated periods, and serum was then obtained from the well and subjected to ELISA analysis. Each panel shows a typical example of the results. Values represent mean ± S.E.M., n = 5, *P<0.05 vs. control. **P<0.05 vs. stimulation with LPS alone. B, D. Effects of CUX1 and sh-CUX1 overexpression on IL-6 protein expression in THP-1 cells and monocytes stimulated with a combination of LPS and either sCD26/DPPIV(+) or sCD26/DPPIV(−). THP-1 cells (5×105) or monocytes (5×105) were incubated in culture medium with or without LPS, with or without various concentrations of sCD26/DPPIV(+) or sCD26/DPPIV(−), with or without CUX1 or sh-CUX1 overexpression, and with or without the DPPIV inhibitor sitagliptin (1 µM). The cells were cultured for the indicated periods, and serum was then obtained from the well and subjected to ELISA. Each panel shows a typical example of the results. Values represent mean ± S.E.M., n = 5, *P<0.05 vs. control. **P<0.05 vs. stimulation with LPS alone. Statistical analysis was performed using Student’s t-test.