The N-terminal region of serum amyloid A3 protein activates NF-κB and up-regulates MUC2 mucin mRNA expression in mouse colonic epithelial cells

Serum amyloid A (SAA) is the major acute-phase protein and a precursor of amyloid A (AA) in AA amyloidosis in humans and animals. SAA isoforms have been identified in a wide variety of animals, such as SAA1, SAA2, SAA3, and SAA4 in mouse. Although the biological functions of SAA isoforms are not completely understood, recent studies have suggested that SAA3 plays a role in host defense. Expression of SAA3 is increased on the mouse colon surface in the presence of microbiota in vivo, and it increases mRNA expression of mucin 2 (MUC2) in murine colonic epithelial cells in vitro, which constitutes a protective mucus barrier in the intestinal tract. In this study, to identify responsible regions in SAA3 for MUC2 expression, recombinant murine SAA1 (rSAA1), rSAA3, and rSAA1/3, a chimera protein constructed with mature SAA1 (amino acids 1–36) and SAA3 (amino acids 37–103), and vice versa for rSAA3/1, were added to murine colonic epithelial CMT-93 cells, and the mRNA expressions of MUC2 and cytokines were measured. Inhibition assays with NF-κB inhibitor or TLR4/MD2 inhibitor were also performed. Up-regulation of MUC2 mRNA expression was strongly stimulated by rSAA3 and rSAA3/1, but not by rSAA1 or rSAA1/3. Moreover, NF-κB and TLR4/MD2 inhibitors suppressed the increase of MUC2 mRNA expression. These results suggest that the major responsible region for MUC2 expression exists in amino acids 1–36 of SAA3, and that up-regulations of MUC2 expression by SAA3 and SAA3/1 are involved with activation of NF-κB via the TLR4/MD2 complex.


Introduction
Serum amyloid A (SAA) is the major acute-phase protein in humans, most mammals, and avians [1]. SAA is also known as a precursor protein of amyloid A (AA) in AA amyloidosis, which is a long-term complication of several chronic inflammatory disorders such as a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 rheumatoid arthritis and juvenile inflammatory arthritis [2]. Differences in amino acid sequence have indicated the existence of multiple SAA isoforms, such as SAA1, 2, 3, and 4 in mouse [1]. SAA1 and SAA2 are well known as main acute-phase isoforms, which are mainly expressed in the liver [1]. SAA3, which is up-regulated during acute and chronic inflammatory responses, is predominantly expressed by macrophages and other cells, including adipocytes, epithelial cells, and endothelial cells in mice [3][4][5]. A fourth isoform, SAA4, is constitutively expressed in the liver [6]. In addition to the difference in primary synthesis site, SAA3 is unique among SAA family members. Among the four SAA isoforms, SAA1, 2, and 4, but not SAA3, have been shown to be associated with high density lipoprotein in mice [7]. Moreover, SAA1 (GenBank accession no. BC087933) and SAA2 (M11130) genes share 95.1% and 92.6% sequence identities in 369 nucleotides and 122 amino acids, respectively, whereas respective identities between SAA1 and SAA3 (NM011315) are 74.3% and 64.7%.
Although the biological functions of SAA isoforms are not completely understood, recent studies have suggested that SAA may play a role in host defense. Shah et al. [8] reported that SAA1 binds to outer membrane protein A of Escherichia coli and Pseudomonas aeruginosa for opsonization, and suggested that SAAs play a role in innate immunity by opsonization of gram-negative bacteria. However, the expression of SAA3, but not SAA1 or 2, is increased on the mouse colon surface in the presence of microbiota [5], and lipopolysaccharide (LPS) strongly induces mRNA expression of SAA3 in murine colonic epithelial CMT-93 cells [5,9,10]. Moreover, our previous study demonstrated that SAA3, but not SAA1, increases mRNA expression of mucin 2 (MUC2) in CMT-93 cells [10]. MUC2 is a high molecular weight gelforming glycoprotein that is secreted into the gut lumen and forms the major mucin component of the protective mucus barrier in the intestinal tract [11]. These results suggest that SAA3 stimulated by LPS relates to intestinal immunity. However, the differences between SAA3 and other SAAs are not fully understood. The mechanism for the induction of MUC2 expression by SAA3 also remains unclear.
In this study, to identify the responsible amino acid sequence region of SAA3 for MUC2 expression, recombinant murine SAA1 (rSAA1), rSAA3, and rSAA1/3, a chimera protein constructed with mature SAA1 (amino acids 1-36) and SAA3 (amino acids 37-103), and vice versa for rSAA3/1, were added to murine colonic epithelial CMT-93 cells, and the mRNA expressions of MUC2 and cytokines were analyzed. Moreover, inhibition assays using NF-κB inhibitor and toll-like receptor 4 (TLR4)/MD2 inhibitor were performed. We demonstrated that MUC2 mRNA expression was significantly up-regulated by rSAA3 and rSAA3/1 compared with rSAA1 and rSAA1/3. In addition, both NF-κB inhibitor and TLR4/MD2 inhibitor suppressed MUC2 mRNA expression by rSAA3 and rSAA3/1, respectively. These results suggest that the major responsible region for MUC2 expression exists in amino acids 1-36 of SAA3, and that up-regulation of MUC2 expression by SAA3 is involved with the activation of NF-κB via the TLR4/MD2 complex.

Expression and purification of rSAA
After confirmation of their sequences, the plasmids were transformed into E. coli BL21 (DE3) pLysS (Invitrogen). Cultured E. coli in Magic Media (Invitrogen) was collected and rSAAs were extracted and purified as described in detail previously [10]. Tag protein from the pRSET A vector was also expressed and purified. Coomassie brilliant blue (CBB) staining and Western blotting (WB) analysis were performed as described previously [10]. Peroxidase activity in WB was visualized by an LAS 4000mini (Fujifilm, Tokyo, Japan).

Quantitative real-time PCR
CMT-93 cells were seeded at 4-6×10 5 cells in 6-well plates and incubated for 15±1 h before experiment. CMT-93 cells were treated with rSAAs at 37˚C for 2 h, washed with PBS, and total RNA was extracted immediately using an RNeasy Mini kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. As for inhibitor assays, tumor necrosis factor (TNF)-α inhibitor (Enzo Life Sciences, Lausen, Switzerland); NF-κB inhibitor, CAPE (Calbiochem, EMD Chemicals, San Diego, CA); or TLR4/MD2 inhibitor, TAK-242 (MedChem Express, Monmouth Junction, NJ) was added to cells at 37˚C for 1 h before incubation with rSAAs. Isolated RNA was quantified using a spectrophotometer GeneQuant 100 (GE Healthcare) and stored at -80˚C until use. Contaminating DNA was removed with DNase I (Invitrogen), and cDNA was synthesized using the SuperScript III First-Strand Synthesis System SuperMix for qRT-PCR (Invitrogen) according to the manufacturer's instructions. Quantitative real-time PCR was performed using a Fast SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) [10]. To investigate mRNA expressions of mucin 2 (MUC2), TNF-α, interleukin (IL)-6, inhibitor κB (IκB)-α, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), their specific primers [12][13][14][15][16][17] were used for real-time PCR (S1 Table). Regenerating islet-derived 3 (REG III)-γ, α-defensin (Def), β-Def-3, and β-Def-4 are anti-bacterial proteins secreted by intestinal epithelial cells by sensing bacteria and bacterial antigens as well as mucins, and contribute to the innate immunity of the intestine [18,19]. Therefore, mRNAs of REG III-γ, α-Def, β-Def-3, and β-Def-4 were also examined by quantitative real-time PCR. Results were normalized to the expression of GAPDH mRNA as an endogenous gene and fold-change relative to control levels were determined by the ΔΔC t method [20]. For verification of specific amplification, a melting-curve analysis of amplification products was performed at the end of each PCR reaction. All experiments were replicated at least three times.

Measurement of cytokines in cell culture supernatant
CMT-93 cells were seeded at 1.2×10 5 cells in 24-well plates and incubated for 15±1 h before experiments. After incubation, CMT-93 cells were treated with rSAAs at 37˚C for 24 h. The

Statistical analyses
The data were collected from at least three independent experiments, expressed as means ± SD, and analyzed for statistical significance by unpaired t-tests.

Induction of cytokine mRNA and protein expressions by rSAAs
Our previous study showed that both SAA1 and SAA3 enhanced IL-6 and TNF-α mRNA expression [10]. To confirm that rSAA1/3 and rSAA3/1 induce cytokines, CMT-93 cells were treated with rSAA1, rSAA3, rSAA1/3, or rSAA3/1, and mRNA and protein expressions of inflammatory cytokines were estimated. There was little difference in IL-6 mRNA expression among the rSAA treatments ( Fig 3A). On the other hand, rSAA1, rSAA3, and rSAA3/1 enhanced TNF-α mRNA expression. In particular, rSAA3 and rSAA3/1 intensively induced TNF-α. Although IL-6 and TNF-α proteins were induced by rSAA3 and rSAA3/1, other cytokines were not induced (Table 1). We therefore confirmed that both IL-6 and TNF-α were induced by rSAA3 and rSAA3/1 at the protein level.
Moreover, to examine whether TNF-α induced by rSAA3 affected the induction of MUC2 expression, cells were exposed to TNF-α inhibitor with rSAA3. TNF-α inhibitor did not significantly affect the induction of MUC2 mRNA expression (Fig 3B), meaning that TNF-α might not contribute to the induction of MUC2 mRNA expression at 2 h.

Induction of IκB-α mRNA expression by rSAAs
It has been reported that SAA3 is an endogenous peptide ligand for the TLR4/MD2 complex, the activated NF-κB signaling pathway in metastatic mouse lung [3], and the TLR4/MD2 complex expressed in colonic epithelial cells [5]. Therefore, we considered that SAA proteins may induce MUC2 mRNA expression through the NF-κB signaling pathway. To test whether the NF-κB signaling pathway was activated by SAAs, cells were treated with rSAA1, rSAA3, rSAA1/3, or rSAA3/1, and then IκB-α mRNA expressions were examined, because IκB-α mRNA levels quantitatively result in NF-κB activation [21]. IκB-α mRNA expressions were strongly induced by rSAA3 and rSAA3/1 (Fig 4); these results were consistent with those obtained from MUC2 mRNA expressions (Fig 2).

NF-κB inhibitor reduced MUC2, TNF-α, and IL-6 mRNA
To test whether SAA proteins regulate MUC2 expression through the NF-κB signaling pathway, cells were exposed to an NF-κB inhibitor, CAPE, before incubation with rSAAs. The NF-κB inhibitor reduced MUC2 mRNA expression by rSAAs (Fig 5). Similarly, the NF-κB inhibitor reduced IL-6 and TNF-α mRNA expressions by rSAAs. These results suggest that rSAA proteins can activate the NF-κB signaling pathway to up-regulate the expressions of MUC2, TNF-α and IL-6 mRNA.
Since it has been reported that TNF-α up-regulates MUC2 expression in human intestinal cancer LS180 cells [23] and colonic epithelial HT-29 cells [24], it is possible that the induction   of MUC2 mRNA expression observed in this study is not an effect of SAA3, but is rather due to the effect of TNF-α induced by rSAA3 because TNF-α also enhances NF-κB independently of the TLR4/MD2-NF-κB signaling pathway. However, inhibition assays revealed that a TNFα inhibitor did not affect the induction of MUC2 mRNA expression, suggesting that TNF-α is not necessary for MUC2 mRNA expression by SAA3. In addition to the up-regulation of MUC2 expression by SAA3 and TNF-α, it has been reported that TNF-α induces SAA3 mRNA expression in CMT-93 cells [5] and mouse granulosa tumor OV3121-1 cells [25]. Moreover, IL-6 induces other mucins, MUC4 and MUC5B, aside from MUC2 [23,26], and also induces SAA3 expression [27][28][29]. These results suggest that MUC2 and other mucins are consecutively produced in cooperation with SAA3 and cytokines, such as TNF-α and IL-6, and that SAA3 plays a role in intestinal immunity with cytokines to protect epithelial cells from bacterial infection. In summary, this study showed that amino acids 1-36 of SAA3 induced MUC2 expression, and we propose a mechanism by which SAA3 induces MUC2 expression in CMT-93 cells after Gram negative bacterial infection (Fig 7). Interestingly, the TFLK motif in bovine mammaryassociated SAA3 increases MUC3 expression in a heterologous host, human intestinal epithelial HT-29 cells [30], a finding that indicates a potential therapeutic/probiotic use of SAA3 to protect intestines from bacterial infection in humans and animals. Further investigations are needed to clarify the essential amino acid sequence of SAA3 for MUC2 expression and to understand the role of SAA in host intestinal immunity in detail.
Supporting information S1