Proton pump inhibitors exert anti-allergic effects by reducing TCTP secretion

Abstract

Background: Extracellular translationally controlled tumor protein (TCTP) is known to play a role in human allergic responses. TCTP has been identified outside of macrophages, in activated mononuclear cells, and in biological fluids from allergic patients. Even TCTP devoid of signal sequences, is secreted to extracellular environment by an yet undefined mechanism. This study is aimed at understanding the mechanism of TCTP release and its regulation. A secondary goal is to see if inhibitors of TCTP release can serve as potential anti-allergic asthmatic drugs.

Methodology/principal findings: Using Western blotting assay in HEK293 and U937 cells, we found that TCTP secretion is reduced by omeprazole and pantoprazole, both of which are proton pump inhibitors. We then transfected HEK293 cells with proton pump expression vectors to search for the effects of exogeneously overexpressed H(+)/K(+)-ATPase on the TCTP secretion. Based on these in vitro data we checked the in vivo effects of pantoprazole in a murine model of ovalbumin-induced allergy. Omeprazole and pantoprazole reduced TCTP secretion from HEK293 and U937 cells in a concentration-dependent fashion and the secretion of TCTP from HEK293 cells increased when they over-expressed H(+)/K(+)-ATPase. In a murine model of ovalbumin-induced allergy, pretreatment with pantoprazole reduced infiltration of inflammatory cells, increased goblet cells, and increased TCTP secretion induced by OVA challenge.

Conclusion: Since Omeprazole and pantoprazole decrease the secretion of TCTP which is associated with the development of allergic reaction, they may have the potential to serve as anti-allergic (asthmatic) drugs.

Figure 1. TCTP secretion, both constitutive and induced from U-937 cells is inhibited by omeprazole and pantoprazole.

(A) TCTP secretion is increased with incubation time in serum-free conditioned media. β-tubulin is a loading control in cells, while it is a cell lysis marker in media. WB: anti-β-tubulin Ab (rabbit polyclonal antibody, H-235, Santa Cruz Biotechnology) and anti-TCTP Ab (rabbit polyclonal antiserum, generated using recombinant His-tagged TCTP as the immunogen), (B) Omeprazole treatment during incubation in conditioned media (5 h) is capable of decreasing of TCTP secretion in concentration-dependent manner. ‘O’ in the graph means omeprazole. WB: anti-TCTP Ab, (C) Pantoprazole treatment (5 h) inhibits TCTP secretion in concentration-dependent manner. ‘P’ in the graph means pantoprazole. WB: anti-TCTP Ab, (D) Both of omeprazole (O) and pantoprazole (P) pre-treatments reduce the TCTP secretion induced by ionomycin (IM) treatment (2 µM, 10 min). 1 µM omeprazole and 100 µM pantoprazole were used. Both chemicals were treated 1 h prior to ionomycin treatment. WB: anti-TCTP Ab. Data from panel B to panel D represent means±S.D. from three independent experiments. #: inhibition, p<0.05, vs. non-treated cells (panel B, C), vs. IM-treated cells (panel D), ##: inhibition, p<0.01, vs. non-treated cells (panel B, C), vs. IM-treated cells (panel D), **: increase, p<0.01, vs. non-treated cells.

Figure 2. The secretion of exogeneously expressed TCTP in HEK293 cells is inhibited by omeprazole and pantoprazole.

(A) TCTP secretion is increased with incubation time in serum-free conditioned media. β-tubulin is a loading control in cells, while it is a cell lysis marker in media. WB: anti-β-tubulin Ab and anti-flag Ab, (B) Omeprazole treatment during incubation in conditioned media (3 h) is capable of decreasing of TCTP secretion in concentration-dependent manner. ‘O’ in the graph means omeprazole. WB: anti-flag Ab, (C) Pantoprazole treatment (3 h) inhibits TCTP secretion in concentration-dependent manner. ‘P’ in the graph means pantoprazole. WB: anti-flag Ab, (D) Both of omeprazole (O) and pantoprazole (P) pre-treatments reduce the TCTP secretion induced ionomycin (IM) treatment (2 µM, 10 min). 1 µM omeprazole and 100 µM pantoprazole were used. Both chemicals were treated 1 h prior to ionomycin treatment. WB: anti-flag Ab. Data from panel B to panel D represent means±S.D. from three independent experiments. #: inhibition, p<0.05, vs. non-treated cells (panel B, C), vs. IM-treated cells (panel D), ##: inhibition, p<0.01, vs. non-treated cells (panel B, C), vs. IM-treated cells (panel D), **: increase, p<0.01, vs. non-treated cells.

Figure 3. Increased TCTP secretion by over-expression of proton pump is inhibited by pantoprazole in HEK293.

(A) Comparison of protein expressions in control and Hαβ samples. Control sample was transfected with two empty vectors (pEGFP-N1 and pcDNAI-neo) and TCTP-3Xflag construct. Hαβ sample was transfected with rat H⁺/K⁺-ATPase α1-GFP, HA-rat Na⁺/K⁺-ATPase β1, and TCTP-3Xflag constructs. WB: anti-GFP Ab (purified rabbit polyclonal antibody, InVitrogen), anti-HA Ab (mouse 12CA5 monoclonal antibody, Santa Cruz), and anti-flag Ab, (B) TCTP secretion is increased by over-expression of H⁺/K⁺-ATPase α1 and Na⁺/K⁺-ATPase β1. Both groups of cells were incubated for 3 h in conditioned media. WB: anti-flag Ab, (C) The secretion assay data for the cells transfected with H⁺/K⁺-ATPase α1, Na⁺/K⁺-ATPase β1, and TCTP. Pantoprazole (1 mM) or omeprazole (1 mM) was treated during 3 h secretion assay. ‘O’ in the graph means omeprazole and ‘P’ does pantoprazole. WB: anti-flag Ab. Data of panel B and panel C represent means±S.D. from three independent experiments. #: inhibition, p<0.05, vs. non-treated cells, *: increase, p<0.05, vs. the cells transfected with two empty vectors (pEGFP-N1 and pcDNAI-neo) and TCTP-3Xflag construct.

Figure 4. Pantoprazole alleviates asthmatic responses and inhibits TCTP secretion in OVA-sensitized/challenged mice.

(A) PAS staining results of lung sections. OVA-sensitized/challenged mice show marked increase of the mucus-producing granular goblet cells in the bronchial alveoli relative to sham mice. The increased PAS positive goblet cells in OVA challenged mice were reduced by pantoprazole pre-treatment in amount dependent manner. Pantoprazole pre-treatment also reduced marked infiltration of the airway wall by PAS positive inflammatory cells, (B) Western blotting data using BALF of allergic mouse model. WB: anti-TCTP antibody. Mice were sensitized with OVA and challenged with PBS or OVA five times every other day. ‘P’ means pantoprazole and indicated numbers (10 and 100) mean dosage. #: inhibition, p<0.05, vs. OVA challenged but no pre-treatment, ##: inhibition, p<0.01, vs. OVA challenged but no pre-treatment, **: increase, p<0.01, vs. sham group. sham: negative control, OVA sensitized but not challenged, n = 4. OVA: positive control, OVA sensitized and challenged with no pre-treatment, n = 5. P 10: OVA sensitized and challenged with pantoprazole 10 mg/kg pre-treatment (30 min), n = 6. P 100: OVA sensitized and challenged with pantoprazole 100 mg/kg pre-treatment (30 min), n = 6.