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. 2021 Apr 20;9(4):879.
doi: 10.3390/microorganisms9040879.

Activation of the JNK/MAPK Signaling Pathway by TGF-β1 Enhances Neonatal Fc Receptor Expression and IgG Transcytosis

Shaoju Qian  1 , Chenxi Li  1 , Xi Liu  1 , Xiangchao Jia  1 , Yuncai Xiao  1   2 , Zili Li  1   2   3
Affiliations

Activation of the JNK/MAPK Signaling Pathway by TGF-β1 Enhances Neonatal Fc Receptor Expression and IgG Transcytosis

Shaoju Qian et al. Microorganisms. .

Abstract

The neonatal Fc receptor (FcRn) transports maternal immunoglobulin G (IgG) to the foetus or newborn and protects the IgG from degradation. FcRn is expressed in several porcine tissues and cell types and its expression levels are regulated by immune and inflammatory events. IPEC-J2 cells are porcine intestinal columnar epithelial cells that were isolated from neonatal piglet mid-jejunum. We hypothesized that transforming growth factor β1 (TGF-β1) upregulated pFcRn expression in IPEC-J2 cells. To test this hypothesis, we treated IPEC-J2 cells with TGF-β1 and demonstrated that porcine FcRn (pFcRn) expression was significantly increased. SP600125, a specific mitogen-activated protein kinase (MAPK) inhibitor, reduced TGF-β1-induced pFcRn expression in IPEC-J2 cells. We performed luciferase reporter assays and showed that the c-JUN sensitive region of the pFcRn promoter gene was located between positions -1215 and -140. The c-JUN sequence, in combination with the pFcRn promoter, regulated luciferase reporter activity in response to TGF-β1 stimulation. Chromatin immunoprecipitation confirmed that there were three c-JUN binding sites in the pFcRn promoter. Furthermore, in addition to increased pFcRn expression, TGF-β1 also enhanced IgG transcytosis in IPEC-J2 cells. In summary, our data showed that the modulation of JNK/MAPK signaling by TGF-β1 was sufficient to upregulate pFcRn expression.

Keywords: JNK pathway; TGF-β1; mucosal immunity; neonatal Fc receptor.

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Conflict of interest statement

The authors have no financial conflict of interest.

Figures

Figure 1
Figure 1
TGF-β1 upregulates pFcRn expression in a dose- and time-dependent manner. (A) IPEC-J2 cells were stimulated with TGF-β1 at the indicated dosages (0, 2, 4, 8 and 16 ng/mL) and pFcRn expression was analysed by Western blotting. (B) IPEC-J2 cells were incubated with TGF-β1 (8 ng/mL) and collected at 1, 2 and 4 h, followed by the Western blot analysis of pFcRn expression. The right panel represents protein band quantification determined by densitometry and normalized to GAPDH; * p < 0.05, ** p < 0.01.
Figure 2
Figure 2
TGF-β1 upregulates pFcRn expression via the JNK/MAPK and c-JUN signaling pathways. (A) IPEC-J2 cells were pre-treated with SP600125 (1, 5, and 10 μM), followed by incubation with TGF-β1 (8 ng/mL) for 12 h. IPEC-J2 cells were harvested and Western blotting was performed as described in Section 2. (B–D) were performed as described in (A) using the following inhibitors: p38 inhibitor SB203580 (1, 5 and 10 μM), ERK inhibitor U0126 (1, 5, and 10 μM), and JNK inhibitor SP600125 (1, 5, and 10 μM). GAPDH was used as a loading control. The right panel represents protein band quantification determined by densitometry and normalized to GAPDH, ** p < 0.01.
Figure 3
Figure 3
Construction of pFcRn promoter luciferase reporter plasmids. (A) Schematic diagram of the pFcRn promoter region and luciferase reporter plasmid. (B) IPEC-J2 cells were co-transfected with a series of truncated pFcRn promoter constructs (−1740 to +350, F1 to F9) and luciferase reporter vector (pRL-TK-luc), and its luciferase activity was measured. (C) IPEC-J2 cells were co-transfected with pFcRn luciferase reporter plasmids and the pRL-TK-luc vector, incubated with TGF-β1 (8 ng/mL) for 12 h, and luciferase activity was quantified; * p < 0.05, ** p < 0.01.
Figure 4
Figure 4
Evaluation of c-JUN binding to porcine pFcRn promoter in vivo. (A) The putative c-JUN binding sites in the pFcRn gene are indicated. Numbers show the putative c-JUN binding sites associated with the transcription start site of the pFcRn gene. TGANRYA, N is A or G, Y is any nucleotide, and Y is A, G, or C. (B) c-JUN components are present in the pFcRn promoter in vivo in response to TGF-β1 treatment. IPEC-J2 cells were incubated with TGF-β1 (8 ng/mL) for 30 min. The ChIP assay was performed using c-JUN-specific Abs (lane 2). IgG was used as the negative control (lane 3). The DNA fragments were analysed by PCR using the primers specified in Table 1. The ChIP assay was repeated at least three times.
Figure 5
Figure 5
Effect of TGF-β1 stimulation on IgG bidirectional transcytosis. A, apical; B, basolateral. IPEC-J2 cells were cultured on 12-well Transwell plates (0.4 μm pore size) for 6–7 days, until TEER > 1000 Ω/cm2. IPEC-J2 cells were incubated with or without TGF-β1 (8 ng/mL) for 12 h. Porcine biotin-IgG or biotin-IgY were added to the apical chamber (lanes 2 and 3) or basolateral chamber (lanes 4 and 5) and incubated for 3 h at 37 °C. Lane 1 corresponds to IgG, ** p < 0.01.
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