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. 2015 Dec 21:13:136.
doi: 10.1186/s12958-015-0132-y.

Regulation of TIMP-1 in Human Placenta and Fetal Membranes by lipopolysaccharide and demethylating agent 5-aza-2'-deoxycytidine

Affiliations

Regulation of TIMP-1 in Human Placenta and Fetal Membranes by lipopolysaccharide and demethylating agent 5-aza-2'-deoxycytidine

Zoë L Vincent et al. Reprod Biol Endocrinol. .

Abstract

Background: An appropriate transcriptional profile in the placenta and fetal membranes is required for successful pregnancy; any variations may lead to inappropriate timing of birth. Epigenetic regulation through reversible modification of chromatin has emerged as a fundamental mechanism for the control of gene expression in a range of biological systems and can be modified by pharmacological intervention, thus providing novel therapeutic avenues. TIMP-1 is an endogenous inhibitor of MMPs, and hence is intimately involved in maintaining the integrity of the fetal membranes until labor.

Objective and methods: To determine if TIMP-1 is regulated by DNA methylation in gestational tissues we employed an in vitro model in which gestational tissue explants were treated with demethylating agent 5-aza-2'-deoxycytidine (AZA) and lipopolysaccharide (LPS).

Results: Quantitative Real-Time PCR (qRT-PCR) revealed that TIMP-1 transcription was significantly increased by combined treatment of AZA and LPS, but not LPS alone, in villous, amnion and choriodecidua explants after 24 and 48 hrs, whilst western blotting showed protein production was stimulated after 24 hrs only. Upon interrogation of the TIMP-1 promoter using Sequenom EpiTyper MassARRAY, we discovered sex-specific differential methylation, in part explained by x-linked methylation in females. Increased TIMP-1 in the presence of LPS was potentiated by AZA treatment, signifying that a change in chromatin structure, but not in DNA methylation at the promoter region, is required for transcriptional activators to access the promoter region of TIMP-1.

Conclusions: Collectively, these observations support a potential role for pharmacological agents that modify chromatin structure to be utilized in the therapeutic targeting of TIMP-1 to prevent premature rupture of the fetal membranes in an infectious setting.

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Figures

Fig. 1
Fig. 1
Schematic of the TIMP-1 gene promoter and locations of primers used for methylation analysis. Region A is upstream of the transcription start site (-275/-1) and covers 11 CpGs, Region B (+1/+279) covers the TIMP-1 promoter and first exon and contains 14 CpGs. The numbered line represents the distance in base pairs from the transcription start site, shown by the number 0. CpGs are represented by open circles
Fig. 2
Fig. 2
TIMP-1 mRNA Expression in Term Villous Placenta, Amnion and Choriodecidua measured by qRT-PCR. a Tissues collected prior to the onset of labor from elective Caesarean sections deliveries (CS, n = 14) are shown by the white bars. Tissues collected following the spontaneous onset of labor from vaginal deliveries (SVD, n = 10) are shown by grey bars. TIMP-1 transcription was further analysed by sex of the fetus in b CS deliveries (Male n = 8, Female n = 6) and c SVD (Male n = 5 and Female n = 5). Data are presented as mean ± SEM * P ≤ 0.05. All results were normalised to the expression of endogenous controls (RPLPO and RPL13a)
Fig. 3
Fig. 3
TIMP-1 mRNA expression as fold change compared to controls in gestational tissues measured using qRT-PCR. Tissues were cultured in the presence of lipopolysaccharide (LPS, 5 μg/ml) with/without prior treatment with 5-aza-deoxycytidine (AZA, 5 μM; n = 8). a Villous placenta b Amnion and c Choriodecidua, and further analysed by the sex of the fetus in d Villous placenta e Amnion and f Choriodecidua. Tissues from pregnancies with male fetuses (n = 5) are shown by white bars and those from pregnancies with female fetuses (n = 3) are shown by cross-hatched bars. Data are calculated as fold change compared to time matched DMSO treated control, represented by the dotted line, and presented as mean ± SEM * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. All results were normalised to the expression of endogenous controls (RPLPO and RPL13a)
Fig. 4
Fig. 4
TIMP-1 protein measured by western blotting in treated explant samples shown as fold change compared to controls. Tissues were cultured in the presence of lipopolysaccharide (LPS, 5 μg/ml) with/without prior treatment with 5-aza-deoxycytidine (AZA, 5 μM; n = 8). a Villous placenta b Amnion and c Choriodecidua and further analysed by the sex of the fetus in d Villous placenta e Amnion and f Choriodecidua. Tissues from pregnancies with male fetuses (n = 5) are shown by white bars and those from pregnancies with female fetuses (n = 3) are shown by cross-hatched bars. Data are calculated as fold change compared to time matched DMSO treated control, represented by the dotted line, and presented as mean ± SEM * P ≤ 0.05. All results were normalised to beta actin optical density
Fig. 5
Fig. 5
Percentage methylated CpGs in the TIMP-1 Promoter in term villous placenta, amnion and choriodecidua collected prior to, or post labor and delivery. a Tissues collected prior to the onset of labor from elective Caesarean sections deliveries (CS, n = 14) are shown by the white boxes. Tissues collected following the spontaneous onset of labor from vaginal deliveries (SVD, n = 10) are shown by grey boxes. TIMP-1 methylation further analysed by sex of the fetus in b CS deliveries (Male n = 8, Female n = 6) and c SVD (Male n = 5 and Female n = 5). Tissues from pregnancies with male fetuses are shown by white or grey bars and those from pregnancies with female fetuses are shown by cross-hatched bars. d Representative epigrams show the % of 5-mC for each tissue at each CpG site within the amplicon for Region B. Percentage methylation calculated from the ratio of mass signals between methylated and non-methylated DNA in each sample. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Data are presented as mean %5-mC + SEM
Fig. 6
Fig. 6
Percentage methylated CpGs in the TIMP-1 promoter in gestational tissues cultured in the presence of lipopolysaccharide (LPS, 5 μg/ml) with/without prior treatment with 5-aza-deoxycytidine (AZA, 5 μM) (n = 8). a Villous placenta; b Amnion and c Choriodecidua. Tissues from pregnancies with male fetuses (n = 5) are shown by white bars and those from pregnancies with female fetuses (n = 3) are shown by cross-hatched bars. For graphical representation, all CpGs within region A have been combined. Representative epigrams for both male and female tissues are shown alongside each graph. * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.001. Percentage methylation calculated from the ratio of mass signals between methylated and non-methylated DNA in each sample. Data are presented as mean %5-mC + SEM

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