KLF5 regulates infection- and inflammation-induced pro-labour mediators in human myometrium

Abstract

The transcription factor Kruppel-like factor 5 (KLF5) has been shown to associate with nuclear factor kappa B (NFkB) to regulate genes involved in inflammation. However, there are no studies on the expression and regulation of KLF5 in the processes of human labour and delivery. Thus, the aims of this study were to determine the effect of i) human labour on KLF5 expression in both foetal membranes and myometrium; ii) the pro-inflammatory cytokine interleukin 1 beta (IL1b), bacterial product flagellin and the viral dsRNA analogue poly(I:C) on KLF5 expression and iii) KLF5 knockdown by siRNA in human myometrial primary cells on pro-inflammatory and pro-labour mediators. In foetal membranes, there was no effect of term or preterm labour on KLF5 expression. In myometrium, the term labour was associated with an increase in nuclear KLF5 protein expression. Moreover, KLF5 expression was also increased in myometrial cells treated with IL1b, flagellin or poly(IC), likely factors contributing to preterm birth. KLF5 silencing in myometrial cells significantly decreased IL1b-induced cytokine expression (IL6 and IL8 mRNA expression and release), COX2 mRNA expression, and subsequent release of prostaglandins PGE 2 and PGF 2a . KLF5 silencing also significantly reduced flagellin-and poly(I:C)-induced IL6 and IL8 mRNA expression. Lastly, IL1b-, flagellin-and poly(I:C)-stimulated NFkB transcriptional activity was significantly suppressed in KLF5-knockout myometrial cells. In conclusion, this study describes novel data in which KLF5 is increased in labouring myometrium, and KLF5 silencing decreased inflammation-and infection-induced pro-labour mediators.

The Transcription Factor Interferon Regulatory Factor-1 (IRF1) Plays a Key Role in the Terminal Effector Pathways of Human Preterm Labor1

“…To determine the expression of IRF1 in fetal membranes and myometrium, immunohistochemistry (IHC) was performed on paraffin sections as described previously [36] using the IHC Select Horseradish Peroxidase Detection Set (Merck Millipore). Briefly, sections were deparaffinized followed by an antigen retrieval step (boiled in 10 mM Tris and 1 mM ethylenediaminetetraacetic acid, pH 9.0, for 10 min followed by 20 min incubation) and then endogenous peroxidases were inactivated by adding 3% hydrogen peroxide for 10 min.…”

Hepatitis A virus cellular receptor 2 (HAVCR2) is decreased with viral infection and regulates pro‐labour mediators OA

“…IHC was performed on paraffin sections as described previously 19 using the IHC Select ® HRP Detection Set (Merck Millipore; Billerica, MA, USA). Briefly, sections were deparaffinized followed by an antigen retrieval step (boiled in 10 mmol/L Tris, 1 mM EDTA, pH 9.0 for 10-min followed by 20-min incubation) and then endogenous peroxidases were inactivated by adding 3% hydrogen peroxide for 10 minutes.…”

RAF1 is increased in labouring myometrium and modulates inflammation-induced pro-labour mediators

“…For the siRAF1 studies, transfection of primary myometrial cells was performed, as we have previously described (Lappas 2015). Briefly, cells at w50% confluence were transfected using Lipofectamine 3000, according to manufacturer's guidelines (Life Technologies).…”

“…Cell viability was assessed by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) proliferation assay, as we have previously described (Lim et al 2014). The response to IL1B and TNF between patients varied greatly, as we have previously reported (Lappas 2015). Thus, data are presented as fold change in expression relative to the expression level in the IL1B-or TNF-stimulated siCONT-transfected cells, which was set at one.…”

“…To determine the expression of RAF1 in myometrium, IHC was performed on paraffin sections, as described previously (Lappas 2015) using the IHC Select HRP Detection Set (Merck Millipore). Briefly, sections were deparaffinised followed by an antigen retrieval step (boiled in 10 mM Tris, 1 mM EDTA, pH 9.0 for 10 min, followed by 20 min incubation) and then endogenous peroxidases were inactivated by adding 3% hydrogen peroxide for 10 min.…”