Human neuronal cells (NT2-N) express functional substance P and neurokinin-1 receptor coupled to MIP-1 beta expression - PubMed (original) (raw)

Human neuronal cells (NT2-N) express functional substance P and neurokinin-1 receptor coupled to MIP-1 beta expression

Yuan Li et al. J Neurosci Res. 2003.

Abstract

Substance P (SP), the most extensively studied and potent member of the tachykinin family, is a major modulator of inflammation and immunomodulatory activities within the central and peripheral nervous systems. We have examined the gene expression of SP and its receptor in a human neuronal cell line (NT2-N). Using reverse transcribed polymerase chain reaction (RT-PCR), the four isoforms of preprotachykinin-A gene transcripts (alpha, beta, gamma, and delta) were detected in the NT2-N. We also identified the presence of mRNA for neurokinin-1 receptor (NK-1R), a primary receptor for SP, in the NT2-N cells. Concomitant with NT2 cell differentiation into neurons, SP and NK-1R mRNA expression increased consistently. Intracellular SP and cell membrane NK-1R immunoreactivity were all observed in NT2-N cells. Most importantly, we demonstrated that SP and NK-1R presented in NT2-N cells are functionally involved in the regulation of macrophage inflammatory protein 1 beta (MIP-1beta), an important beta-chemokine participating in the activation and directional migration of immune cells to sites of central nervous systems (CNS) inflammation. Thus, SP and its receptor may play an important role in modulation of neuronal functions related to regulation of immune activities within the CNS. The NT2-N cell line is well suited for in vitro investigations of the SP-NK-1R pathway in immune responses and inflammation in the CNS.

Copyright 2002 Wiley-Liss, Inc.

PubMed Disclaimer

Figures

Fig. 1

Fig. 1

Reverse transcribed-polymerase chain reaction (RT-PCR) amplification of preprotachykinin-A (PPT-A) gene transcript (A) with the primer pair of HSP4/HSP3 and neurokinin-1 receptor (NK-1R) gene transcript (B) of human neuronal cells (NT2-N; 4 weeks after retinoic acid [RA] treatment). Sizes are estimated from DNA Ladder (100-bp fragments) coelectrophoresesed as markers. Marker,100-bp fragments of DNA ladder; −, negative control in which template was omitted; +, human brain tissue as positive control; NT2-N; RT-PCR-amplified PPT-A transcript (A) or NK-1R gene transcript (B) of NT2-N.

Fig. 2

Fig. 2

RT-PCR amplification of PPT-A transcripts of NT2-N (4 weeks after RA treatment) with the primer pairs of HSP4/HSP67 and HSP4/HSP7. Sizes are estimated from DNA Ladder (100-bp fragments) coelectrophoresesed as markers. Marker, 100-bp fragments of DNA ladder as markers; NT2-N, RT-PCR-amplified PPT-A transcripts of NT2-N using primer pairs of HSP4/HSP67 (β and γ isoforms) or HSP4/HSP7 (α and δ isoforms), respectively. RNA transcripts derived from α and δ, γ, and β isoform-containing plasmids are used as positive controls as indicated.

Fig. 3

Fig. 3

RT-PCR analysis and real-time RT-PCR quantification of Substance P (SP) gene expression during NT2 differentiation into NT2-N. RA-treated NT2 cells at different time points as indicated were selected to evaluate SP mRNA expression and quantification by RT-PCR (inset) and real-time RT-PCR, respectively. The SP mRNA levels were normalized on the basis of the SP mRNA/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA ratio obtained by the real-time RT-PCR.

Fig. 4

Fig. 4

RT-PCR analysis and real-time RT-PCR quantification of NK-1R mRNA expression during NT2 differentiation into NT2-N. The same RNA samples used in Figure 3 were also analyzed for NK-1R gene expression by RT-PCR (inset) and real-time RT-PCR (SYBR Green). The NK-1R mRNA levels were normalized on the basis of the NK-1R mRNA/GAPDH mRNA ratio obtained by the real-time RT-PCR.

Fig. 5

Fig. 5

Immunohistochemical staining of the intracellular SP in the NT2-N cells using anti-SP antibody. The NT2-N cells (4 weeks after RA treatment) were stained with either rabbit anti-human SP antiserum (A) or normal rabbit serum (B). The magnification is ×400.

Fig. 6

Fig. 6

Immunofluorescent staining of the membrane SP receptor (NK-1R) on the NT2-N cells using anti-NK-1R antibody. The NT2-N cells (4 weeks after RA treatment) were stained with either a rabbit anti-human NK-1R antibody (A) or normal rabbit IgG (B). The magnification is ×400.

Fig. 7

Fig. 7

Effect of SP on macrophage inflammatory protein 1 beta (MIP-1β) expression in NT2-N cells. A: Effect of SP on MIP-1β mRNA expression. NT2-N cells were incubated with SP (10−8 M to 10−6 M) for 3 hr and total RNA was isolated and subjected to RT-PCR and electrophoresis (inset) and real-time PCR to quantify MIP-1β mRNA. The quantitative data are expressed as mean of triplicate cultures of MIP-1β mRNA copy number per 103 copies of GAPDH mRNA. B: Effect of SP on MIP-1β protein production in NT2-N cells. NT2-N cells were incubated with SP (10−8 M to 10−6 M) for 24 hr, MIP-1β protein in the supernatants were then assayed using enzyme-linked immunosorbent assay (ELISA). The data shown are presented as the mean ± S.D. of triplicate cultures and are representative of three independent experiments (*P < 0.05).

Similar articles

Cited by

References

    1. Andrews PW. Retinoic acid induces neuronal differentiation of a cloned human embryonal carcinoma cell line in vitro. Dev Biol. 1984;103:285–293. - PubMed
    1. Beczkowska IW, Gracy KN, Pickel VM, Inturrisi CE. Inducible expression of N-methyl-D-aspartate receptor, and delta and mu opioid receptor messenger RNAs and protein in the NT2-N human cell line. Neuroscience. 1997;79:855–862. - PubMed
    1. Black IB, Adler JE, Dreyfus CF, Jonakait GM, Katz DM, LaGamma EF, Markey KM. Neurotransmitter plasticity at the molecular level. Science. 1984;225:1266–1270. - PubMed
    1. Bozic CR, Lu B, Hopken UE, Gerard C, Gerard NP. Neurogenic amplification of immune complex inflammation. Science. 1996;273:1722–1725. - PubMed
    1. Carter MS, Krause JE. Structure, expression, and some regulatory mechanisms of the rat preprotachykinin gene encoding substance P, neurokinin A, neuropeptide K, and neuropeptide gamma. J Neurosci. 1990;10:2203–2214. - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources