Hippocampal Cholinergic Neurostimulating Peptide Suppresses LPS-Induced Expression of Inflammatory Enzymes in Human Macrophages

Abstract

Hippocampal cholinergic neurostimulating peptide (HCNP) is a secreted undecapeptide produced through proteolytic cleavage of its precursor protein, HCNPpp. Within hippocampal neurons, HCNP increases gene expression of choline acetyltransferase (ChAT), which catalyzes acetylcholine (ACh) synthesis, thereby modulating neural activity. HCNPpp also appears to be expressed in various immune cells. In the present study, we observed that HCNPpp is expressed in U937 human macrophage-like cells and that HCNP exposure suppresses lipopolysaccharide (LPS)-induced gene expression of ChAT. The opposite action is also seen in T lymphocytes, which suggest that HCNP appear to suppress cholinergic system in immune cells. In addition, HCNP suppresses LPS-induced gene expression of inflammatory enzymes including cyclooxygenase 2 (COX2) and inducible nitric oxide (NO) synthase (iNOS). The suppressive effect of HCNP may reflect suppression of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling activated by LPS. Thus, HCNP may have therapeutic potential as an anti-inflammatory drug.

INTRODUCTION

Hippocampal cholinergic neurostimulating peptide (HCNP) is an undecapeptide generated by cleavage of the N-terminal of HCNP precursor protein (HCNPpp).^1,2) HCNP was originally identified in hippocampal neurons, where it induces expression of choline acetyltransferase (ChAT) to increase the production of acetylcholine (ACh), leading to maturation of the neurons.^1,2) Notably, decreased expression of HCNP and its precursor has been seen in the brains of patients with Alzheimer’s disease,³⁾ suggesting that HCNP is regulated physiologically and pathologically, and that it affects neural activity through ACh synthesis. HCNPpp is also reportedly expressed in a variety of other cells and tissues.^4,5) For example, Goumon et al. reported that HCNP is released together with adrenaline from adrenal chromaffin cells and that it acts on M₂ muscarinic ACh receptors (mAChRs) in the heart, mediating a negative inotropic effect under basal conditions and counteracting adrenergically induced positive inotropism.⁶⁾

Immune cells express both ChAT and AChRs, which constitute a non-neuronal cholinergic system that modulates immune and inflammatory function.^7,8) We previously reported that T cells and other immune cells express HCNPpp, and that long-term exposure to HCNP reduces the ACh content of T cells by reducing ChAT gene expression.⁹⁾ Here, we assessed expression of HCNPpp in U937 human macrophage-like cells and investigated the effects of HCNP on resting cells and on cells activated using lipopolysaccharide (LPS).

MATERIALS AND METHODS

Cell Culture

U937 human macrophage-like cells were cultured in RPMI 1640 containing 7% fetal bovine serum (FBS), 100 units/mL penicillin and 100 µg/mL streptomycin at 37°C under a humidified atmosphere with 5% CO₂.

Materials and Antibodies

Synthetic HCNP was purchased from Bachem (Bubendorf, Basel-Landschaft, Switzerland). LPS was from Sigma-Aldrich (St. Louis, MO, U.S.A.). Anti-extracellular signal-regulated kinase (ERK), anti-phosphorylated ERK, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and anti-cyclooxygenase (COX)2 antibodies were all from Cell Signaling Technology (Danvers, MA, U.S.A.).

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western Blotting

U937 cells were lysed in 20 mM Tris–HCl (pH 7.4) with 2% SDS. Phosphatase inhibitor cocktail (Nacalai Tesque, Kyoto, Japan) was added to the lysis buffer to prevent dephosphorylation of ERK. The lysates were then subjected to 4–12% Bis-Tris SDS-PAGE (Thermo Fisher Scientific, Waltham, MA, U.S.A.), after which the separated proteins were transferred to nitrocellulose membranes (Thermo Fisher Scientific). The membranes were blocked using Blocking One (Nacalai Tesque) for 1 h at room temperature and then incubated first with primary antibodies at 4°C overnight and then with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h. The blots were developed using SuperSignal West Pico Chemiluminescent substrate (Thermo Fisher Scientific), after which the chemiluminescence was detected using an ECL system (Amersham Imager 600, Cytiva, Marlborough, MA, U.S.A.).

Real-Time PCR

Total RNAs were extracted from U937 cells using Sepasol RNA II Super (Nacalai Tesque), and cDNAs were prepared by reverse transcription using a Prime Script RT reagent Kit (TaKaRa Bio, Kusatsu, Shiga, Japan) with a S1000 Thermal Cycler (Bio-rad, Hercules, CA, U.S.A.). Real-time PCR analysis was conducted using CYBR premix EX taq, FAM-labeled probes, and predesigned primers (TaKaRa Bio) with a Thermal Cycler Dice Real Time System (TaKaRa Bio). The primer pairs were as follows: for ChAT (HA221706) 5′-AGC CCT GCC GTG ATC TTT G-3′ and 5′-GCA CAG TCA GTG GGA ATG GAG T-3′; for HCNPpp (HA243336) 5′-CCA GCA GGA AGG ATC CCA AAT-3′ and 5′-CTG CTC GTA AAC CAG CCA GAC A-3′; for COX2 (HA293877) 5′-AGC CTG AAT GTG CCA TAA GAC TGA-3′ and 5′-AAA CCC ACA GTG CTT GAC ACA GA-3′; inducible nitric oxide (NO) synthase (iNOS) (HA282449) 5′-CAG CAA GCA GCA GAA TGA GTC C-3′ and 5′-TGC ATC CAG CTT GAC CAG AGA-3′; GAPDH (HA067812) 5′-GCA CCG TCA AGG CTG AGA AC-3′ and 5′-TGG TGA AGA CGC CAG TGG A-3′.

Statistical Analysis

Data are presented as means ± standard error of the mean (S.E.M.). All representative experiments were repeated three times. Statistical analysis was performed using SigmaPlot (Systat Software Inc., San Jose, CA, U.S.A.). Differences between two groups were evaluated using Student’s t-test, and among three or more groups using one- and two-way ANOVA with post hoc Dunnett’s or Tukey’s test, respectively. Values of p < 0.05 were considered significant.

RESULTS AND DISCUSSION

Real-time PCR analysis revealed that HCNPpp in expressed in U937 macrophage-like cells to the same degree as in MOLT3 human T lymphoid cells (Fig. 1). This suggests that HCNP may be released from U937 cells after proteolytic cleavage of HCNPpp.

Fig. 1. Expression of HCNPpp mRNA in MOLT3 Human Leukemic T Cells and U937 Human Macrophage Cells

Expression of HCNPpp mRNA was assessed in MOLT3 and U937 cells using real-time PCR. HCNPpp mRNA levels were normalized to GAPDH mRNA. Means ± S.E.M. (n = 3).

In MOLT3 cells, long-term exposure to HCNP results in a decreased levels of ChAT expression.⁹⁾ In U937 cells, no ChAT expression was seen under resting conditions, but it was dramatically increased in response to 1 µg/mL LPS (Fig. 2A). HCNP did not affect ChAT mRNA levels in U937 cells under resting conditions, but it significantly suppressed LPS-induced ChAT gene expression (Fig. 2A). This suggests that, as in T cells, HCNP negatively regulates cholinergic signaling in U937 macrophages by suppressing ChAT gene expression. Because macrophages activated by LPS via Toll-like receptor 4 (TLR4) release several pro-inflammatory cytokines and mediators, including prostanoids and NO, we next investigated whether HCNP exposure affects expression of two inducible enzymes, COX-2 and iNOS, in U937 cells. We found that HCNP suppressed LPS-induced COX2 and iNOS gene expression, though HCNP had no effect on their expression in unstimulated cells (Fig. 2B). Correspondingly, HCNP also decreased LPS-induced expression of COX2 protein (Fig. 2C).

Fig. 2. HCNP Suppresses LPS-Induced ChAT, COX2 and iNOS Expression in U937 Cells

A. ChAT mRNA levels. U937 cells were exposed to 1 µg/mL LPS for 24 h in the absence or presence of 300 pg/mL HCNP. ChAT mRNA levels were assessed using real-time PCR. Bars depict means ± S.E.M. (n = 3). *** p < 0.001. B. COX2 and iNOS mRNA levels. Bars depict means ± S.E.M. (n = 3). *** p < 0.001. C. COX2 protein levels. GAPDH was used as a loading control. The graph shows the relative expression level of COX2 protein. COX2 was quantified using densitometry and normalized to GAPDH. Bars depict means ± S.E.M. (n = 3). *** p < 0.001.

HCNP activates myocardial M₂ mAChRs, thereby exerting a negative inotropic effect under basal conditions and counteracting adrenergically induced positive inotropism.^6,10) However, scopolamine, a mAChR antagonist, did not inhibit HCNP-induced suppression of ChAT and COX2 gene expression (Fig. 3A). This suggests HCNP does not act via mAChRs in macrophages.

Fig. 3. HCNP Suppresses LPS-Induced ERK Phosphorylation in U937 Cells

A. Effect of mAChR blockade on HCNP-induced suppression of ChAT and COX2 gene expression. U937 cells were exposed to 1 µg/mL LPS for 24 h in the absence or presence of 300 pg/mL HCNP with and without 10 µM scopolamine. Bars depict means ± S.E.M. (n = 3). *** p < 0.001. B. Levels of ERK phosphorylation in U937 cells. U937 cells were exposed to 1 µg/mL LPS for the indicated times in the absence or presence of 300 pg/mL HCNP. Right: Graph showing the phosphorylated ERK/ERK ratio. Bars depict means ± S.E.M. (n = 3). *** p < 0.001 vs. Control.

Because LPS-induced TLR4 activation leads in turn to activation (phosphorylation) of ERK, we tested whether HCNP inhibits TLR4 signaling. We found that HCNP suppressed LPS-induced ERK phosphorylation in U937 cells (Fig. 3B). Thus, HCNP may prevent LPS-induced macrophage activation by inhibiting activity in the TLR pathway.

In this study, we revealed that HCNP suppresses ChAT gene expression in macrophages. Similar results were obtained from T cells,⁹⁾ suggesting that HCNP negatively regulates cholinergic signaling in immune cells. On the contrary, HCNP increases ChAT expression in hippocampal neurons, resulting in the increased ACh production.²⁾ Several species of ChAT mRNAs transcribed from three distinct promoters and produced by alternative splicing of noncoding exons are reportedly expressed in human.¹¹⁾ The transcriptional regulation of ChAT gene with different promoters might account for the opposite response to HCNP between immune cells and neurons.

Macrophages release several cytokines and inflammatory mediators in response to LPS. Our results suggest that HCNP suppresses induction of related inflammatory mediators, including iNOS and COX2. Although HCNP reportedly acts as a M₂ mAChR agonist that suppresses the inotropic effect of adrenergic agonists on cardiac function,^6,10) we found that M₂ mAChRs are not involved in HCNP-mediated suppression of COX2 and ChAT gene expression. Instead, the effect may reflect suppression of signaling triggered by TLR4 activation. HCNP is generated by cleaving the N-terminus of HCNPpp, which is alternatively referred to as Raf kinase inhibitory protein (PEBP) and reportedly inhibits the mitogen-activated protein kinase (MAPK)/ERK by interacting with Raf-1 and mitogen-activated protein extracellular kinase (MEK), as well as the nuclear factor-kappaB (NFκB) pathway.^2,12,13) Our findings suggest HCNP may retain some of the functionality of HCNPpp. Alternatively, HCNP may act as an antagonist preventing LPS binding to TLR4. In addition, we revealed that HCNP also suppresses ChAT gene expression in macrophages. Similar results were obtained from T cells,⁹⁾ suggesting that HCNP negatively regulates cholinergic signaling in immune cells. Macrophages express muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively).^7,8) Non-neuronal cholinergic system is involved in the regulation of immune and inflammatory responses. Especially, in macrophages, G_q/11-coupled mAChR activation appear to enhance the release of cytokines such as tumor necrosis factor-α (TNF- α) after exposure to LPS.^14,15) Thereby, Decreased ChAT gene expression by HCNP might reduce ACh production and release in immune cells, which partially and indirectly suppress LPS-induced gene expression of inflammatory enzymes. Determining where in the pathway which HCNP acts will be the aim of a future study.

In summary, our findings suggest that HCNP acts as an immunosuppressant in macrophages, providing possible evidence of the therapeutic potential of HCNP.

Acknowledgments

This study was supported in part by a Grant-in-Aid for Scientific Research (C) (18K06903) from the Ministry of Education, Science, Sports and Culture of Japan, and by Individual Research Grants from the Doshisha Women’s College of Liberal Arts (19-31TF).

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Ojika K, Kojima S, Ueki Y, Fukushima N, Hayashi K, Yamamoto M. Purification and structural analysis of hippocampal cholinergic neurostimulating peptide. Brain Res., 572, 164–171 (1992).
• 2) Ojika K, Mitake S, Tohdoh N, Appel SH, Otsuka Y, Katada E, Matsukawa N. Hippocampal cholinergic neurostimulating peptides (HCNP). Prog. Neurobiol., 60, 37–83 (2000).
• 3) Maki M, Matsukawa N, Yuasa H, Otsuka Y, Yamamoto T, Akatsu H, Okamoto T, Ueda R, Ojika K. Decreased expression of hippocampal cholinergic neurostimulating peptide precursor protein mRNA in the hippocampus in Alzheimer disease. J. Neuropathol. Exp. Neurol., 61, 176–185 (2002).
• 4) Katada E, Mitake S, Matsukawa N, Otsuka Y, Tsugu Y, Fujimori O, Ojika K. Distribution of hippocampal cholinergic neurostimulating peptide (HCNP)-like immunoreactivity in organs and tissues of young Wistar rats. Histochem. Cell Biol., 105, 43–51 (1996).
• 5) Saunders PT, McKinnell C, Millar MR, Gaughan J, Turner KJ, Jegou B, Syed V, Sharpe RM. Phosphatidylethanolamine binding protein is an abundant secretory product of haploid testicular germ cells in the rat. Mol. Cell. Endocrinol., 107, 221–230 (1995).
• 6) Goumon Y, Angelone T, Schoentgen F, Chasserot-Golaz S, Almas B, Fukami MM, Langley K, Welters ID, Tota B, Aunis D, Metz-Boutigue MH. The hippocampal cholinergic neurostimulating peptide, the N-terminal fragment of the secreted phosphatidylethanolamine-binding protein, possesses a new biological activity on cardiac physiology. J. Biol. Chem., 279, 13054–13064 (2004).
• 7) Fujii T, Mashimo M, Moriwaki Y, Misawa H, Ono S, Horiguchi K, Kawashima K. Physiological functions of the cholinergic system in immune cells. J. Pharmacol. Sci., 134, 1–21 (2017).
• 8) Fujii T, Mashimo M, Moriwaki Y, Misawa H, Ono S, Horiguchi K, Kawashima K. Expression and function of the cholinergic system in immune cells. Front. Immunol., 8, 1085 (2017).
• 9) Saito Y, Mashimo M, Nobeyama A, Murakami K, Fujii T. Hippocampal cholinergic neurostimulating peptide suppresses acetylcholine synthesis in T lymphocytes. Biol. Pharm. Bull., 41, 1611–1614 (2018).
• 10) Angelone T, Goumon Y, Cerra MC, Metz-Boutigue MH, Aunis D, Tota B. The emerging cardioinhibitory role of the hippocampal cholinergic neurostimulating peptide. J. Pharmacol. Exp. Ther., 318, 336–344 (2006).
• 11) Misawa H, Matsuura J, Oda Y, Takahashi R, Deguchi T. Human choline acetyltransferase mRNAs with different 5′-region produce a 69-kDa major translation product. Brain Res. Mol. Brain Res., 44, 323–333 (1997).
• 12) Hengst U, Albrecht H, Hess D, Monard D. The phosphatidylethanolamine-binding protein is the prototype of a novel family of serine protease inhibitors. J. Biol. Chem., 276, 535–540 (2001).
• 13) Vallée BS, Coadou G, Labbé H, Sy D, Vovelle F, Schoentgen F. Peptides corresponding to the N- and C-terminal parts of PEBP are well-structured in solution: new insights into their possible interaction with partners in vivo. J. Pept. Res., 61, 47–57 (2003).
• 14) Xu ZP, Yang K, Xu GN, Zhu L, Hou LN, Zhang WH, Chen HZ, Cui YY. Role of M3 mAChR in in vivo and in vitro models of LPS-induced inflammatory response. Int. Immunopharmacol., 14, 320–327 (2012).
• 15) Wang Z, Li M, Liu L, Geng B. Muscarinic M1 and M2 receptor subtypes play opposite roles in LPS-induced septic shock. Pharmacol. Rep., 71, 1108–1114 (2019).