NF-κB regulates a cassette of immune/inflammatory genes in human pregnant myometrium at term

Abstract

The onset of human labour resembles inflammation with increased synthesis of prostaglandins and cytokines. There is evidence from rodent models for an important role for nuclear factor-κB (NF-κB) activity in myometrium which both up-regulates contraction-associated proteins and antagonizes the relaxatory effects of progesterone. Here we show that in the human, although there are no differences in expression of NF-κB p65, or IκB-α between upper- or lower-segment myometrium or before or after labour, there is nuclear localization of serine-256-phospho-p65 and serine-536-phospho-p65 in both upper- and lower-segment myometrium both before and after the onset of labour at term. This shows that NF-κB is active in both upper and lower segment prior to the onset of labour at term. To identify the range of genes regulated by NF-κB we overexpressed p65 in myocytes in culture. This led to NF-κB activation identical to that seen following interleukin (IL)-1β stimulation, including phosphorylation and nuclear translocation of p65 and p50. cDNA microarray analysis showed that NF-κB increased expression of 38 genes principally related to immunity and inflammation. IL-1β stimulation also resulted in an increase in the expression of the same genes. Transfection with siRNA against p65 abolished the response to IL-1β proving a central role for NF-κB. We conclude that NF-κB is active in myocytes in both the upper and lower segment of the uterus prior to the onset of labour at term and principally regulates a group of immune/inflammation associated genes, demonstrating that myocytes can act as immune as well as contractile cells.

Fig 4

Overexpression of NF-κB p65 increases the nuclear localization and phosphorylation of both p65 and p50. Myometrial cells were either transfected with p65 or stimulated with IL-1β (1 ng/mL) for 30 min. Nonstimulated cells transfected with empty vector were used as control. Separate nuclear and cytoplasmic protein fractions were used for Western analysis for serine-536-phospho-p65 (A, B), serine-337-phospho-p50 (C, D), NF-κB p65 (E, F), NF-κB p50 (G, H). To confirm nuclear/cytoplamsic separation Western analysis was performed on the same proteins for α-tubulin (cytosol specific) and Lamin b (nuclear specific) (I, J). Results were normalized against the nuclear and cytoplasmic controls α-tubulin and lamin-b respectively and are expressed as mean ± S.E.M. (n= 3). RT-PCR data showed an increase in NF-κB p65 48 hrs following transfection of a p65 expression vector (K) and are expressed on a log scale as mean ± S.E.M. (n= 6). A time course study shows that p65 protein overexpression is maximal as early as 24 hrs but sustained for at least 48 hrs (L).

Fig 1

No labour-associated or regional differences in NF-κB proteins in myometrium at term. Whole-cell tissue protein was extracted from matched upper and lower-segment myometrial biopsies collected before (L−) or after (L+) labour onset (example Western blots n=2 for each tissue type, bar graphs total n=4 for each tissue type). Western analysis was performed for IkBa (A; P= 0.9938 by ANOVA), p65 (B; P= 0.4177 by ANOVA), and Ser536-P-p65 (C; P= 0.4414 by ANOVA). Blots were scanned for densitometric analysis and values normalized against densitometry values for Gβ or GAPDH.

Fig 2

No labour-associated or regional differences in expression or nuclear localization of p65 in myometrium at term. Paraffin-embedded tissue taken from matched upper and lower-segment myometrial biopsies collected prior to (L−) or following (L+) labour onset was subject to immunofluorescent analysis, using p65 with red-fluorescent Alexa Fluor 594 dye and a-smooth muscle actin (α-sm-actin) was visualized with green-fluorescent Alexa Fluor 488 dye. Cell nuclei were visualized with DAPI (blue). (L−, n=5, L+, n=6). Objective lens used was x40.

Fig 3

No labour-associated or regional differences in serine 276 phosphorylation of p65 in myometrium at term. Immunofluorescent analysis of serine 276-phosphorlyated p65 (Ser276-P-p65) expression in paraffin-embedded matched upper (US) and lower (LS) segment myometrial biopsies taken at term either prior (L−) or following (L+) labour onset. Ser276-P-p65 was visualized with red-fluorescent Alexa Fluor 594 dye and a-smooth muscle actin (α-sm-actin) was visualized with green-fluorescent Alexa Fluor 488 dye. Cell nuclei were visualized with DAPI (blue). (L−, n=3, L+, n=6). Objective lens used was x63.

Fig 5

Overexpression of NF-κB p65 increases the DNA binding of both p65 and p50 to levels that are comparable to IL-1β stimulation. A 100-fold excess of unlabelled κB cold (NF-κB oligo) or irrelevant (oct-1) oligonucleotide were added before the addition of 32P-labelled probe as DNA binding competitor to verify specificity of binding. NF-κB Nuclear proteins were supershifted with both p50 (lanes 4, 9) and p65 antibodies (lanes 5, 10).

Fig 6

Effect of IL-1β, overexpression of p65 and knockdown of p65 on mRNA expression of selected genes. Myometrial cells were incubated with IL-1 (1 ng/ml) for 24 hrs after which total RNA was extracted and mRNA expression of selected genes was measured by qPCR. Note the logarithmic scale. (A) Mean ± S.E.M. n= 6, *P < 0.05 Wilcoxon matched pairs analysis. Myometrial cells were transfected with the expression construct for p65. The empty expression vector pSG5 was used as control. After 24 hrs, total RNA was extracted and mRNA expression of selected genes was measured by qPCR. Note the logarithmic scale. (B) Mean ± S.E.M. n= 7, *P < 0.05 Wilcoxon matched pairs analysis. Myometrial cells were transfected with either siRNA for p65 or a non-targeting siRNA control. After 72 hrs cells were incubated with IL-1 (1 ng/ml) for a further 24 hrs, after which total RNA was extracted and mRNA expression of selected genes was measured by qPCR. Note the linear scale. (C) Mean ± S.E.M. n= 6, *P < 0.05 Wilcoxon matched pairs analysis.

Fig 7

siRNA in myometrial cells effectively knocks down NF-κB p65 protein expression. Myometrial cells were transfected with either NF-κB p65 expression vector or siRNA against NF-κB p65. Non targeting siRNA was used as control. Western analysis of myometrial whole cell lysate was performed for NF-κB p65 after 72 hours incubation.