Neuromedin B and Its Receptor Influence the Activity of Myometrial Primary Cells In Vitro Through Regulation of Il6 Expression via the Rela/p65 Pathway in Mice1

Abstract

The neuromedin B receptor (Nmbr) is an important physiological regulator of spontaneous activities and stress responses through different cascades as well as its autocrine and paracrine effects. Previous studies have revealed that neuromedin B (Nmb) and its receptor signal via the Rela (also known as p65)/Il6 pathway in a mouse model of pregnancy. This study investigated the mechanism of Nmbr signaling via the Rela/p65-Il6 pathway and regulation of the concentration of intracellular free calcium ([Ca(2+)](i)) during the onset of labor in primary mouse myometrial cell cultures isolated from mice in term labor. Data demonstrated Nmbr agonist-mediated upregulation of the DNA binding activity of Rela/p65, Il6 expression, and [Ca(2+)](i) in a concentration-dependent manner. Furthermore, a significant correlation was observed between DNA binding activity of Rela/p65 and Il6 expression. Moreover, this up-regulation was blocked by Nmbr and Rela/p65 knockdown, achieved by RNA interference (RNAi) technology. No significant differences were identified in the inhibition of Il6 expression as a result of Nmbr or Rela/p65 knockdown. However, significant differences were observed between the [Ca(2+)](i) in Rela/p65-specific group and that in the Nmbr-specific small interfering RNA (siRNA)-treated groups. These data demonstrated that the Nmb/Nmbr interaction in pregnant myometrial primary cells in vitro predominantly influenced uterine activity through regulation of Il6 expression via the Rela/p65 pathway, although the effects of Nmbr on [Ca(2+)](i) involved several pathways that remain to be elucidated.

Genomic Analysis of Spontaneous Abortion in Holstein Heifers and Primiparous Cows

“…While research regarding its impact on fetal loss is limited, Zhang and colleagues (2012) investigated Nmb and its receptor's (Nmbr) roles in pre-term pregnancies in mice. The authors suggested that the interaction between Nmb/Nmbr may influence myometrial cells via the Rela/p65 pathway [99]. The Rela/p65 pathway is an important upstream regulator of the labor-induced processes involving uterine smooth muscle through regulation of phospholipase C/protein kinase C, myosin light-chain phosphorylation, and free intracellular calcium [99,100].…”

“…The authors suggested that the interaction between Nmb/Nmbr may influence myometrial cells via the Rela/p65 pathway [99]. The Rela/p65 pathway is an important upstream regulator of the labor-induced processes involving uterine smooth muscle through regulation of phospholipase C/protein kinase C, myosin light-chain phosphorylation, and free intracellular calcium [99,100]. Mutations or dysregulation of this NMB may induce an SA or pre-term birth due to its involvement in regulating genes involved in calcium cascades (NCLAD, CAMK2G, PLCB1, PRKCB) and its previous association with uterine smooth muscle contractions [97,99].…”

Interaction between NF-κB and AP-1 and their intracellular localization at labor in human late pregnant myometrial cells in vivo and in vitro

“…Primary cell cultures have been shown to retain morphological and functional characteristics and expression of NMBR were shown to be retained for at least 4 generations. [ 9 ] This study explored the interactions between NF-κB and AP-1 and their intracellular localization changes the first time in the USMCs to discover the underlying mechanism of labor onset, by using HLPMCs before or after labor onset in vivo and primary culture HLPMCs in vitro. Standard immunochemical staining techniques were used to demonstrate positive expression of a-SMA and NMBR in the primary culture of myometrial cells, thus confirming the myogenic origin of cultured cells.…”

“…Our previous studies have demonstrated that NMB and its receptor can induce labor onset in pregnant mice and influence the activity of myometrial primary cells in vitro via a RELA (NF-κBp65)/IL6-mediated pathway. [ 8 , 9 ] Those findings strongly indicated that NMB binding to NMBR can activate both NF-κB and AP-1 pathways to induce labor initiation, and some interactions between NF-κB and AP-1 may exist to play a synergistic role in the process of parturition initiation. The present study proposed to use co-immunoprecipitation (CO-IP), western blotting, immunohistochemistry (IHC), and immunocytofluorescence (ICF) techniques to explore their interaction and intracellular localization changes in the myometrial cells in vivo and in vitro to discover the underlying mechanism of labor onset.…”

“…[ 6 , 7 ] Previous studies of our group also demonstrated that activation of the RELA (NF-κBp65)/IL6-mediated pathway can induce labor onset in pregnant mice and influence the activity of myometrial primary cells in vitro. [ 8 , 9 ] AP-1, as another main TF in the regulation of inflammatory cytokines, is also receiving increasing attention for its potential role in labor onset. [ 10 , 11 ] It has been confirmed that NF-κB and AP-1could be activated to induce the expression of downstream inflammatory cytokines at the same time when stimulated by lipopolysaccharide (LPS).…”