Glucocorticoid Receptor Regulated KCNA5 Mediates Cell Proliferation in Human Fetal Membranes

doi:10.21203/rs.3.rs-1594481/v1

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Glucocorticoid Receptor Regulated KCNA5 Mediates Cell Proliferation in Human Fetal Membranes

https://doi.org/10.21203/rs.3.rs-1594481/v1

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Background

Preterm birth is a major public health issue, affecting approximately 10% of pregnancies in the United States. A major identifiable cause of preterm birth is preterm premature rupture of membranes (PPROM). PPROM is a pregnancy complication in which the amnion and chorion weaken and rupture prior to 37 weeks of pregnancy and before contractions have begun. PPROM is responsible for 30–40% of preterm birth cases. Recently, PPROM has been closely linked to maternal stress, leading us to hypothesize that glucocorticoid signaling may contribute to PPROM.

Methods

We measured the gene expression effects of glucocorticoids in primary amnion cells using RNA-seq. KCNA5 emerged as a potential GR regulated gene. To measure the effects of KCNA5 on cell proliferation in primary amnion epithelial cells, we used siRNA to reduce KCNA5 expression and Cas9-based epigenome editing to increase KCNA5 expression in HEK293T cells; followed by cellular assays to measure effects of proliferation and apoptosis.

Results

KCNA5 knockdown significantly increased cell proliferation without appearing to impact apoptosis. CRISPR-mediated over expression of endogenous KCNA5 significantly decreased cell proliferation.

Conclusions

Glucocorticoid-mediated activation of KCNA5 contributes to decreased cell proliferation in cultured primary amnion epithelial cells. Decreases in amnion epithelial proliferation could impair the ability of these cells to repair microfractures in the membrane and lead to overall membrane weakening consistent with PPROM.

Glucocorticoids

Amnion

Cell Proliferation

Preterm premature rupture of membranes (PPROM) is a leading cause of preterm births. PPROM is defined be rupture of the fetal membrane rupture before 37 weeks of gestation. PPROM is the leading identifiable cause of preterm birth, accounting for 30–40% of cases [1]. The amnion epithelial layer plays a crucial role in membrane integrity. Amnion epithelial cells are constantly turning over and remodeling [2]. Amnion epithelial proliferation is crucial to wound repair and microfracture repair in the fetal membranes. The amnion epithelial layer proliferates to repopulate itself, and undergoes epithelial mesenchymal transition to repopulate the collagen producing mesenchymal layer beneath it [3].

One contributing factor to all cause preterm birth and to PPROM specifically is maternal psychological stress. Maternal adversity is an independent predictor of preterm births [4–6]. In one study of PPROM specifically, women under psychological stress had 100 times greater risk of PPROM compared to unstressed mothers [7]. Even though the risk for PPROM is high, preventative strategies are limited [8].

A potential mechanism by which maternal stress impacts gestation is via cortisol signaling. Psychological stress increases circulating cortisol, in some cases up to five fold [9]. The effect of elevated glucocorticoid levels long term is counter to expectations. In chronic stress conditions, gene expression changes show reduced glucocorticoid receptor activation and related gene expression and increased pro-inflammatory signaling in response to the same levels of cortisol [10–14].

The interplay of psychological stress and altered cortisol levels has been shown to affect birth timing. Cortisol signaling likely plays an important role in labor initiation as evidenced by the surge in maternal, fetal and amniotic fluid levels of glucocorticoids close to term [15–17]. There is evidence of altered cortisol regulation in pregnant women who have experienced adverse childhood experiences [18, 19]. There is also association between plasma cortisol levels and the recurrence of preterm birth. Specifically, median maternal plasma cortisol levels are elevated in women who have had one isolated preterm birth, and elevated further in women who have had recurrent preterm birth [20]. There is a link between elevated cortisol and maternal depression that is thought to increase risk for preterm birth [21].

Elevated cortisol levels contribute to myriad physiological effects including immunosuppression [22, 23], impaired wound healing [24, 25], and other detrimental effects [26–28]. The causes of impaired wound healing include cortisol-dependent effects on cell proliferation such as cell cycle arrest [24, 25]. Of particular relevance here, glucocorticoid signaling is intact in the amnion [29]. Glucocorticoids have an anti-inflammatory effect in the amnion, the primary weight bearing layer in fetal membranes [30]. Medroxyprogesterone acetate (MPA), known to have glucocorticoid activity, has also been shown to inhibit cytokine induced matrix metalloproteinase and chemokine release in both primary amnion epithelial and mesenchymal cells via the glucocorticoid signaling pathway [30, 31]. Another study showed that in the amnion epithelial cells of fetal membranes, a local increase in glucocorticoids stimulates apoptosis via the tissue-type plasminogen system activation of caspase 3 [32].

At a molecular level, cortisol and related glucocorticoids act by altering gene regulation via the inducible transcription factor activity of the glucocorticoid receptor [33–36]. In this study, we sought to determine the role of glucocorticoid signaling in maintaining fetal membrane integrity. To do so, we investigated glucocorticoid-dependent changes in gene expression in cultured primary amnion epithelial cells. That analysis revealed increased expression of the voltage gated potassium ion channel KCNA5. We further show that KCNA5 impacts proliferation in amnion epithelial cells, suggesting a molecular mechanism by which glucocorticoid signaling can contribute to PPROM.

Primary Amniotic Epithelial Cell Culture

Primary amniotic epithelial cells were isolated from 12 healthy term cesarean section patients for knockdown studies and 10 patients for RNA-seq studies according to Casey and MacDonald 1996 [37]. The collection and use of these samples were approved by the Duke University Institutional Review Board as an exempt protocol for using discarded placentas after schedules normal term cesarean sections. Briefly, the amnion layer was removed from the rest of the membrane with sterile forceps. We rinsed the layer three times in fresh Dulbecco’s Modified Eagle Medium/Nutrient Mixture 12 (DMEM/F12) supplemented with 100x antibiotic-antimycotic supplement containing 10,000 units/mL of penicillin, 10,000 µg/mL of streptomycin, and 25 µg/mL of Gibco Amphotericin B (Thermo Fisher, Waltham, Massachusetts) to remove other cell types. The rinsed amniotic membrane was cut into pieces and incubated in DMEM/F12 media with the same antibiotic-antimycotics as described above and 1 g of Gibco trypsin 1:250 powder (MilliporeSigma, St Louis, Missouri) for 30 minutes at 37°C in a shaking water bath. After 30 minutes, we ran the mixture through a metal strainer to separate disassociated cells in the first digest from the remaining intact membrane. The remaining membrane was placed back in fresh DMEM/F12 trypsin mixture for a second digest. We added DMEM/F12 with 100x Antibiotic-Antimycotic and 10% fetal bovine serum (FBS) (Gibco, Gaithersburg, MD) to the filtered portion and then spun cells at 2000 RCF for 5 minutes. We removed the supernatant and the resuspended pelleted cells in DMEM/F12 with Antibiotic-Antimycotic and 10% FBS. Incubation and shaking with the trypsin solution was repeated with the second digest of the membranes. We combined the cells from the first and second digest and plated on 10 cm tissue culture treated petri dishes. The cells were incubated in humidified air with 5% CO₂ and media was changed every two days. At 95% confluence, approximately a week after initial plating, we passaged the cells using 0.25% trypsin with EDTA (Gibco, Gaithersburg, MD) and plated at approximately 0.5 x 10⁶ cells/mL in 6-well and 96-well tissue culture treated plates for subsequent experiments.

RNA-seq

To measure changes in gene expression, we used primary amnion epithelial cells collected and treated previously [31]. For samples from each of 12 individuals, we treated primary amnion epithelial cells with a control siRNA (siRNA, ID: AM4611; Thermo- Fisher, Waltham, Massachusetts) or glucocorticoid receptor siRNA (ID: AM51331; Thermo Fisher, Waltham, Massachusetts) using Lipofectamine RNAimax (Thermo Fisher, Waltham, Massachusetts). Stock solution of Medroxy Progesterone Acetate (MPA) was made by dissolving solid MPA (MilliporeSigma, St Louis, Missouri) in 200 proof ethanol at a concentration of 10^− 2 M. Then 72 hours after siRNA transfection, we treated the cells for 6 h with 1 mL of stock MPA solution for a final concentration of 10^− 6 M MPA for 6 hours. We added 10 ng/mL Tumor Necrosis Factor (TNFa) challenge (R&D Systems, Minneapolis, Minnesota) to a half of the cells for an additional 24 hours. Cells were lysed with TRIzol reagent (Thermo Fisher, Waltham, Massachusetts). RNA was isolated using the RNeasy mini kit (Qiagen, Hilden, Germany). RNA was quantified with a Qubit fluorometer (Thermo Fisher, Waltham, Massachusetts) and analyzed with an RNA tape station screen tape (Agilent, Santa Clara, California). For each sample, we constructed RNA-seq libraries from 0.5 mg of RNA using the TruSeq stranded mRNA kit following the standard protocol (Illumina, San Diego, California). We sequenced resulting libraries on an Illumina Next seq using 25 bp paired end sequencing.

RNA-seq read alignment and signal estimation

Samples were sequenced to a depth of 7.5 and 215 M. Illumina adapters found in the FASTQ reads were removed using Trimmomatic v0.32 [38]. Reads less than 20 nt after trimming were filtered out from further analysis. Sequences were aligned to GRCh38 human reference genome using the alignment tool STAR v2.4.1a [39] following the a 2-pass strategy to first identify a splice junctions to improve the overall mapping quality. STAR was run with default parameters except for ‘--outFilterMultimapNmax 1’ to remove multi-mapping reads. TPM (transcripts per million) and RPKM (reads per kilobase of transcript per million reads mapped) were computed for each mapped gene using RSEM v1.2.25 [40].

We removed genes with less than 10 reads in at least two libraries. Library size was normalized using trimmed mean of M values (TMM) normalization in the Bioconductor edgeR package [41] within the R statistical programming environment. We fit a linear negative binomial mixed model to the resultant gene matrix with patient identifier set as a random intercept and siRNA and TNFa as factors. We calculated the false discovery rate (FDR) using the Benjamini-Hochberg method. Genes with FDR below 0.05 were considered for follow up analysis.

KCNA5 Knockdown

We cultured primary amnion epithelial cells as described above and split into 6-well or 96-well tissue culture treated plates at 0.5-1.0x10⁶ cell/mL density for 24 hours. We washed cells with serum and antibiotic-antimycotic free DMEM/F12 and then incubated in serum and antibiotic-antimycotic free DMEM/F12. The cells were transfected with control (ID# s7689, Thermo Fisher, Waltham, Massachusetts) or KCNA5 (ID #s7689, Thermo Fisher, Waltham Massachusetts) siRNA using Lipofectamine RNAiMAX (Invitrogen. Carlsbad, CA) and opti-MEM (Gibco, Gaithersburg, Maryland) and the standard RNAiMAX protocol. The final concentration of siRNA was 10 nM in all conditions. Twenty-four hours after lipofection, we added DMEM/F12 media supplemented with 20% FBS to the wells for a final FBS concentration of approximately 8%. We treated the first nine wells per row of the 96 well plate with bromodeoxyuridine. Seventy-two hours post transfection, all cells were harvested for ELISA (described below) or RNA isolation. For RNA isolation, we collected cell lysate with buffer RLT and isolated RNA using the RNeasy mini kit with the optional DNase digestion (Qiagen, Hilden, Germany). We measured the success of the siRNA using real time quantitative polymerase chain reaction (RT-qPCR). We quantified RNA with Qubit fluorometer (Thermo Fisher, Waltham, Massachusetts) and analyzed with RNA tape station screen tape (Agilent, Santa Clara, California). For each sample, we reverse transcribed 100 ng of RNA to cDNA using the Superscript III first strand synthesis system (Invitrogen, Carlsbad, CA) with oligo dT 12–18 (Invitrogen, Carlsbad, CA) as the primer. We used fifty ng of cDNA as a template for real time quantitative polymerase chain reaction (RT-qPCR) using validated TaqMan gene expression probes targeting KCNA5 (Thermo Fisher, Waltham, Massachusetts, assay ID: Hs04991697_s1) and GAPDH (Thermo Fisher, Waltham, Massachusetts, assay ID: Hs02786624_g1) with the Taqman universal PCR master mix (Applied Biosystems, Foster City, California). We performed RT-qPCR using the Applied Biosystems Step One Real Time PCR system (Applied Biosystems, Foster City, California) using the following protocol: initial denaturation and activation of polymerase at 95°C for 10 minutes, then 40 cycles of a two-step amplification process of 95°C for 15 seconds and 60°C for one minute. Selected patient samples were tested, and we evaluated knockdown using the 2^− DDCT method normalizing relative KCNA5 expression to GAPDH.

BrdU ELISA

We cultured primary amnion epithelial cells from 12 patients in a 96 well tissue culture treated plate with 50,000 cells per well. One day after plating, we added KCNA5 siRNA to half the wells and control siRNA to the other half using Lipofectamine RNAiMax to transfect cells. We diluted BrdU stock reagent from Abcam BrdU Cell Proliferation Colorimetric Assay (Abcam, Cambridge, United Kingdom) 1:500 in completed DMEM/F12 media. We added BrdU to the first nine rows of cells fifty-four hours after knockdown to begin to measure cell proliferation. Eighteen hours after the addition of BrdU, we aspirated media from the wells and added 200 mL of fixing solution from the same Abcam BrdU kit to each well. We incubated plates at room temperature for 30 minutes. After 30 minutes, we removed the fixing solution. The plates were stored in a Ziplock bag at 4°C for no more than one month. After 3–4 membranes were collected, we followed the standard Abcam BrdU assay protocol. We measured absorbance at 450 nm using GloMax Discover system (Promega, Madison, Wisconsin). Nine well technical replicates and three well blank measurements from each row were averaged separately. We subtracted the average of the blank measurements from the average of the assay measurements.

Active Caspase 3 ELISA

We measured human active Caspase 3 Ser29 levels using the SimpleStep Elisa Human Caspase 3 ser29 kit from Abcam (Abcam, Cambridge, United Kingdom). We lysed cells in media with provided cell extraction buffer supplemented with Halt 100x proteinase inhibitor cocktail (Thermo Fisher, Waltham, Massachusetts). Lysed cells were stored at -80°C. After 3–4 membranes had been collected, we thawed samples on ice and then we followed the SimpleStep kit protocol with each sample run in duplicate. We measured absorbance at 450 nm using GloMax Discover system (Promega, Madison, Wisconsin). Technical replicates were averaged for downstream analysis.

KCNA5 Overexpression

We designed eight guides using GuideScan [42] for the coordinates annotated as promoters immediately upstream of KCNA5 in the ENCODE candidate cis regulatory element data set [43]. Those guides are listed in Additional File 2. We added selected target sequence and a nontargeting control into the guide RNA template DNA fragment outlined by Mali et al. 2013 [44]. We ordered DNA fragments from Eurofins and cloned them into the Zero Blunt TOPO PCR Cloning Kit (Thermo fisher, Waltham, Massachusetts). We plated bacteria onto LB-agar plates containing 50 mg/mL of kanamycin. We selected single colonies of bacteria and grew them in 100 mL LB with 50 mg/mL kanamycin overnight. We then isolated plasmids using the Machery-Nagel endotoxin free Nucleobond Xtra Midi Kit (Machery Nagel, Düren, Germany). HEK293T cells stably expressing dCas9-P300 (Addgene ID #83889) were gifted from Charles Gersbach [45]. We transfected cells with each guide plasmid using the Lonza SF Cell Line 4D-NucleofectorTM X Kit S (Lonza, Basel, Switzerland). We transfected guides three times in three independent transfections of 400,000 cells each. Each transfection was split into three wells of a 12 well plate. We harvested cells from each individual transfection at 24, 48, and 72 hours post transfection. We isolated RNA from cells using the Qiagen RNeasy 96 Kit (Qiagen Hilden, Germany). One replicate of the 24 hour timepoint for all guides was lost during RNA isolation. We reverse transcribed the RNA into cDNA using Superscript 3 first strand synthesis system (Thermo Fisher, Waltham, Massachusetts). We used TaqMan probes for KCNA5 (Thermo Fisher, Waltham, Massachusetts, assay ID: Hs04991697_s1) and Beta Actin (Thermo Fisher, Waltham, Massachusetts, assay ID: Hs03023943_g1) in qPCR to measure induction. Guide 8 performed the best by qPCR 48 hours post transfection with increases in expression largely maintained through 72 hours post transfection. We then transfected HEK293T cells used above with a nontargeting guide (Additional File 2) or guide 8 using five cuvettes each of Lonza SF Cell Line 4D NucleofectorTM X Kit L (Lonza Basel, Switzerland). We pooled each transfection on the same guide. We plated approximately 20,000 cells per well in a separate 96 well plate filled for each guide. Additionally, we plated 3 wells of a 6 well plate for each guide transfection with approximately 100,000 cells per well. At 48 hours, we added BrdU to the first 9 rows of each 96 well plate. We harvested the cells in the 6 well plate, isolated RNA and made cDNA as described above. We used the same TaqMan probes as described above for qPCR and found an approximately 3.6 cycle difference in Δ CT values as shown in Fig. 5A. Eighteen hours after the addition of BrdU, we fixed the cells and measured cell proliferation as described in the BrdU ELISA above.

Immunohistochemistry

To determine the expression patterns of KCNA5 in fetal membranes, we performed immunohistochemistry staining on four fetal membrane samples. These samples were collected from term pregnant women who were not in labor at cesarean delivery from a site distant to area overlying the cervix as a part of a prior observational study [46]. The collection and use of these samples were approved by the Duke University Institutional Review Board. Briefly, we fixed sections of previously collected fetal membranes in paraffin and prepared slides. We deparaffinized issue sections with xylene followed by graded rehydration in ethanol (100, 95, 80 and 70%) and distilled water. Subsequently, we subjected sections to heat-induced epitope retrieval by heating in antigen unmasking solution (Vector Laboratories, INC, Burlingame, CA) preheated to more than 90°C for 20 minute (two 10 minute periods with reheating between), followed by a 20 minute cool-down period at room temperature. We stained slides using UltraVision LP Detection System HRP Polymer & DAB Plus Chromogen kit following manufacturer’s instruction (Thermo Fisher Scientific Inc, Fremont, CA). This UltraVision detection system detects a specific mouse IgG or rabbit IgG antibody bound to an antigen in tissue sections. The specific antibody is located by a universal secondary antibody formulation conjugated to an enzyme-labeled polymer that recognizes mouse and rabbit immunoglobulins. We then visualized the polymer complex with 3,3’-diaminobenzidine tetrahydrochloride (DAB) substrate. We used KCNA5 antibody from rabbit (catalog No. APC-004, Alomone Labs, Jerusalem, Israel) at 1:200 dilution in PBS with 1% BSA and 5% goat serum on three patient samples. The KCNA5 antibody we used detects the intracellular C-terminus of the protein. For a negative control, we incubated one patient sample with polyclonal rabbit IgG antibody (catalog no. AB27472, Abcam, Cambridge, MA) at a dilution of 1:200. Slides were then counterstained with hemotoxylin and eosin stain and images were photographed using Zeiss Axio Observer.

Glucocorticoid-mediated gene expression responses in the primary amnion epithelial cells

Previous research has shown that medroxyprogesterone acetate (MPA) attenuates the response of primary amnion cells to tumor necrosis factor alpha (TNFa) induced matrix metalloproteinase 9 (MMP9) expression and activity [31]. MMP9 upregulation in fetal membranes is associated with reduced tensile strength [47] and more frequent spontaneous rupture [48]. Allen et al. 2019 [31] demonstrated that the ability of MPA to counteract the pro-inflammatory pathways of TNFa was mediated primarily through the GR in amnion epithelial cells, demonstrating the role of GR in molecular mechanisms that lead to PPROM.

To investigate the broader consequences of MPA-dependent glucocorticoid responses on TNFa challenge in amnion epithelial cells, we measured gene expression changes in primary amnion epithelial samples from ten patients [31] using RNA-seq. To identify GR-dependent effects, we compared MPA responses in cells with and without glucocorticoid receptor knock down, and with or without TNFa challenge. We sequenced the RNA-seq libraries using paired-end 25 bp reads on Illumina sequencing instruments. We sequenced between 7.5 and 215 M reads per sample, and read quality was high for all samples (Additional file 1).

Additional File 1. RNA-seq quality. Reads sequenced and alignment rate for each library are listed as an estimate of library quality.

Most (67%) of the variation in gene expression between samples can be largely attributed to the patient (Fig. 1A). To account for patient variability when estimating the gene expression effects of GR knockdown and TNFα challenge, we used a linear mixed model with the patient identifier as a random effect. That model identified 1041 genes with significantly altered gene expression by GR knockdown compared to a control siRNA in the presence of MPA (Fig. 1B). That result supports the ability of MPA to act through the GR. The genes significantly altered include known glucocorticoid-responsive genes including members of the interleukin and interferon family [49], KLF9 [50], and PER1 [51] (Additional File 2).

We prioritized one gene, KCNA5, for follow up study. KCNA5 was upregulated by GR knockdown (β = 2.9, false discovery rate [FDR] = 0.0002). KCNA5 is a member of the Shaker family of potassium ion channels that have been implicated in cell differentiation and proliferation. The role of KCNA5 regulating cell proliferation has been shown in several cell type. In Ewing sarcoma cells, KCNA5 acts as a cell stress sensor and reduces proliferation in response to DNA damage. When silenced, cells proliferate more rapidly following this damage [52]. Repression of KCNA5 also decreases apoptosis in cell stress conditions in Ewing sarcoma cells [53]. Reducing KCNA5 expression by siRNA in granulosa cells induces proliferation for 72 hours [54]. Conversely, upregulation of KCNA5 increased caspase 3 activity and apoptosis in kidney fibroblast-like cell line COS-7 [55].

Alterations of proliferation and apoptosis could affect the barrier function of the fetal membrane. Amnion epithelial cells increase proliferation in response to injury and microfractures of the membranes. A portion of the proliferating amnion epithelial cells then undergo epithelial mesenchymal transition to repopulate the collagen producing mesenchymal layer beneath it [3]. Disruptions to this process could impair the ability of the amnion to remodel to repair damage and lead to rupture of membranes.

To investigate if glucocorticoid signaling regulates KCNA5 expression in other contexts, we searched for additional examples in datasets deposited in the Gene Expression Omnibus (GEO). One study [56] measured how glucocorticoids, specifically dexamethasone, alter human hemopoietic stem cell differentiation and gene expression. In their study, they treated CD34 + hemopoietic stem cells from three healthy donors with cytokines (FLT3LG, SCF, IL-7, and IL-15) and then treated the cells with 500 nM dexamethasone or DMSO as a vehicle control for the total time in culture, 35 days. In that study, KCNA5 was found to be downregulated by dexamethasone by more than 20-fold (FDR = 4 x 10^− 6).

Distribution of KCNA5 expression in human reproductive tissues

To investigate if there is KCNA5 expression in tissues relevant to PPROM, we searched expression profiles from the Human Protein Atlas [57]. The Human Protein Atlas is a collection of 44 different tissues and organs from 122 patients stained by 24,028 antibodies covering to 16,975 protein-encoding genes. Of those antibodies, 20,456 were produced in house and 3,572 were sourced from external suppliers. Over 13 million immunohistochemistry images were taken and analyzed by pathologists. The Human Protein Atlas includes observations of several female reproductive tissues including placenta. In the Human Protein Atlas, Kv1.5, the protein encoded by KCNA5, was detected at low or medium levels broadly throughout the human body. The Human Protein Atlas assigned an expression level of not detected, low, medium or high based on staining intensity and portion of cells stained. Across female reproductive tissues, there is medium expression of Kv1.5 in the endometrium, and low expression in the vagina, fallopian tube, and placenta. Amnion tissue specifically was not included in this study.

To investigate more specifically the expression of Kv1.5 in the fetal membrane layers, we immunohistochemically stained fetal membranes from three healthy term c-section patients with an antibody raised against the intracellular C-terminus of the Kv1.5 ion channel. We demonstrated that Kv1.5 is expressed in the certain layers of human fetal membranes (Fig. 2, representative image). In the amnion, Kv1.5 was detected in the amnion epithelial cells and primarily localized to the cytoplasm with occasional localization to the nucleus (Fig. 2B). Kv1.5 was also detected in amnion mesenchymal cells in the amnion and cytotrophoblast cells in the chorion layer (Fig. 2B).

Reduced KCNA5 expression increases proliferation in primary amnion epithelial cells but does not affect apoptosis

Based on the above results, we hypothesized that KCNA5 may contribute to PPROM by impacting cell proliferation in the amnion epithelium. As a first step to test that hypothesis, we tested whether reduced KCNA5 expression led to increased proliferation of amnion epithelial cells. To do so, we recruited 19 additional healthy term c-section patients (Fig. 3) and successfully collected primary amnion epithelial cells for knockdown from 12 patients. Gestation length varied from 37 weeks to 42 weeks. Of the 19 samples collected, four did not proliferate enough for studies described below, potentially due to cell senescence in amnion close to parturition [58]. Those samples were excluded from future analysis. We then knocked down KCNA5 expression using siRNA for 72 hours, and measured effects relative to cells from the same patients that were treated with non-targeting siRNAs. In the samples chosen to measure knockdown, we observed an 84% reduction in KCNA5 expression on average (Fig. 4). These samples were chosen based on cell number after passage. There was substantial variability in knockdown efficiency across patients, ranging from an increase of 46% (half cycle difference likely to be noise) to 97% decrease.

Next, to measure the proliferative effects of changes in KCNA5 expression, we used a bromodeoxyuridine (BrdU) assay to measure changes in the amount of newly synthesized DNA when KCNA5 is knockdown. Briefly, BrdU is a synthetic nucleotide that, when included in cell culture media, integrates into newly synthesized DNA. Changes in cell proliferation can then be estimated by measuring differences in the amount of BrdU incorporation over time.

We measured changes in BrdU incorporation using a colorimetric ELISA with and without KCNA5 knockdown in primary amnion epithelial cells. Cells were treated with siRNA for a total of 72 hours, and treated with BrdU for the last 18 hours before harvesting. We first completed a pilot study of three patient samples and used the preliminary results for a power calculation to determine our final population size. We estimated that 11 patient samples total would give us 90% power to detect differences in proliferation due to KCNA5 knockdown. We next collected and assayed KCNA5-dependent proliferation in an additional 9 healthy term c-section patients for a total of 12 patients. We observed increases in proliferation after knockdown in eight out of 12 patients but observed substantial patient specific differences in proliferation. Therefore, to control for patient variability we assessed the effects the KCNA5 knockdown on proliferation relative to a control siRNA in cells from the same patient. We estimated that KCNA5 knockdown resulted in a 19% increase in cell proliferation (Fig. 5A, p = 0.03, two-sided t test).

KCNA5 has been documented to impact both cell proliferation [52–54] and apoptosis [55]. Using the same samples as above, we also tested whether KCNA5 knockdown affected apoptosis. To assess apoptosis in our 12 patient samples, we measured active caspase 3 ser29 levels 72 hours after siRNA transfection, Overall, active caspase levels were low for all patients (Fig. 5B), and we did not observe significant differences in active caspase levels with KCNA5 knockdown (p = 0.547).

Recruiting P300 to the KCNA5 promoter increases expression in human embryonic kidney cells

To understand fully the effect of changes in KCNA5 expression, we tested the converse hypothesis that increased KCNA5 expression reduces cell proliferation. To increase endogenous KCNA5 expression, we used a CRISPR-based epigenome editing system. In that system, catalytically inactive Cas9 (dCas9) is fused to the P300 histone acetyltransferase domain (dCas9-P300) and recruited to target specific sequences via guide RNAs [45, 59]. Targeting dCas9-P300 to gene promoters have been shown to be sufficient to increase expression of a gene in its local chromatin context [59]. Due to a lack of cell lines to model amnion epithelial cells and challenges over expressing KCNA5 in primary amniotic cells, we used a more tractable human embryonic kidney cell line, HEK293T, that was engineered to stably express dCas9-P300 (HEK293T^{dCas9 − P300}).

We designed eight guide RNAs to the KCNA5 promoter using GuideScan [42]. Specifically, we targeted the genomic region immediately upstream of the transcription start site that was marked as a promoter in the ENCODE registry of candidate cis-regulatory elements [43]. Sequences and targets of each individual guide can be found in Additional File 2. To test the effects of each guide RNAs in HEK293T^{dCas9 − P300} cells, We transiently expressed each guide RNA under the control of a U6 promoter [44].

To determine the time after transfection when dCas9-P300 produced the strongest gene expression response, we tested effects at 24, 48, and 72 hours after transfection with each guide RNA (Fig. 6A). The best performing guide had maximum expression of KNCA5 at 48 hours post transfection with high levels of expression remaining at 72 hours post transfection. Expression increased by almost 10-fold from the worst performing guide and timepoint (guide 3 at 72 hours) to the best performing guide and timepoint (guide 8 at 48 hours). We used those best conditions – guide 8 at 48 hours post transfection – for all subsequent experiments,

Increased KCNA5 expression reduces proliferation in human embryonic kidney cells

We next measure the effect of KCNA5 overexpression on cell proliferation. Cells were transfected with the top guide or a non-targeting control (Additional File 2). Forty-eight hours after transfection, RNA was isolated from cells in the 6 well plate to measure KCNA5 induction (Fig. 6C). Cells transfected with the KCNA5 targeting guide had a 12-fold increase in KCNA5 expression compared to cells transfected with the nontargeting guide. At the same time point, BrdU was added to mark newly synthesized DNA and actively proliferating cells. Eighteen hours after the addition of BrdU, cells were fixed to measure relative BrdU. Overexpression of KCNA5 in HEK293T^{dCas9 − P300} cells is associate with 35% decrease in proliferation (p-value = 0.028) (Fig. 6D). The observed decrease in proliferation with KCNA5 overexpression combined with the observed increase in proliferation with reduced KCNA5 expression indicate that KCNA5 expression levels impact cell proliferation.

Many signals are integrated in the maternal and fetal cells leading to parturition, however it is unclear how each signal may affect different tissues involved in gestation. While glucocorticoid activity is known to be altered in parturition [15–17], the effect of changes in its activity in fetal cells remain largely unknown. In this study, we identified an ion channel known to act as a cell stress sensor, KCNA5 [52], as a part of the glucocorticoid response in primary amnion epithelial cells. Understanding the role of this gene could lead to greater understanding of the integration of pro-inflammatory signaling and glucocorticoid signaling in a clinically relevant context.

The relationship between voltage gated potassium channels and glucocorticoid signaling is not fully understood. Mineralocorticoid signaling upregulates protein Kv1.5 levels in atrial monocytes [60]. The mineralocorticoid receptor has similar affinities to mineralocorticoids and glucocorticoids and is activated by cortisol [61]. Additionally, the mineralocorticoid receptor forms heterodimers with the glucocorticoid receptor [62]. Leukocytes, macrophages, and other immune cells express voltage gated potassium channels, including KCNA5. Inflammation alters the activity of these channels, depolarizing the cells, and altering downstream cellular processes, such as proliferation [63]. Obesity induced inflammatory signaling through the NLRP3 inflammasome upregulates KCNA5 expression in atrial tissue [64].

To isolate the effect of KCNA5 specifically and understand its role in the amnion, we used siRNA to knockdown its expression in vitro using primary amnion epithelial cells. In this system, we identified a 19.2% increase in proliferation. This change in proliferation was not accompanied by any compensatory change in apoptosis. Active Caspase 3 levels remained unchanged when KCNA5 was knocked down. One limitation of this study is that caspase independent cell death was not measured. However, the overall effect of caspase independent cell death is unlikely to substantially affect the balance between cell death and division [65]. This one directional change in cell turnover could alter overall cell number over the course of later stages of pregnancy.

Because KCNA5 is repressed by glucocorticoids and via the glucocorticoid receptor, we wanted to study what the consequent effect would be in cells. We used HEK293T cells expressing dCas9-P300 and transfected guides targeting the KCNA5 promoter. This system allowed us to easily target and alter expression of our gene of interest to test its effect on proliferation. By recruiting P300 to the KCNA5 promoter, we were able to significantly increase its expression. KCNA5 overexpression significantly decreased proliferation. This is consistent with its role as a cell stress sensor halting proliferation when conditions are not optimal [52]. We were unable to study overexpression specifically in primary amnion epithelial cells but the effect in those cells of KCNA5 knockdown suggests KCNA5 expression plays a role in the cell cycle for the amnion epithelial layer.

Both the extracellular matrix and amnion epithelial cells contribute to the tensile strength of the amnion [66]. Additionally, amnion epithelial cells are primarily responsible for healing wounds and microfractures within the amnion. Amnion epithelial cells proliferate and undergo epithelial-mesenchyme transition to repopulate both the epithelial layer and the collagen producing mesenchyme layer beneath it [3]. Microfractures in the amnion that remain unhealed are more prevalent in laboring membranes than non-laboring membranes when matched for gestational age [67]. We have found evidence that KCNA5 changes expression in response to glucocorticoid signaling in amnion epithelial cells. These changes in KCNA5 expression alter cellular proliferation and may lead to cell cycle arrest. Physiologically, these changes could reduce the tensile strength of fetal membranes and their ability to heal microfractures and wounds, potentially leading to PPROM. Even in the absence of microfractures, premature arrest in amnion epithelial cell proliferation will likely have an impact on the ability of fetal membrane to remodel in the face of a growing fetus affecting its tensile strength and lead to fetal membrane weakening.

Taken all together, our data suggests that KCNA5 plays a key role in integrating glucocorticoid signaling in the fetal membrane. If cells remain responsive to glucocorticoid signaling, KCNA5 levels remain low enough to allow cell division to occur as normal and the membrane to retain its barrier function. If cells are no longer responsive to glucocorticoids due to long term exposure or chronic stress, KCNA5 expression levels can rise leading to reduced proliferation. This paradigm could highlight the role glucocorticoid receptor signaling plays in maintaining fetal membrane integrity and by extension the role of maternal stress on preterm birth related outcomes

PPROM

Preterm Premature Rupture of Membranes

MPA

Medroxyprogesterone Acetate

Glucocorticoid Receptor

Progesterone Receptor

KCNA5

Potassium Voltage-Gated Channel Subfamily A Member 5

siRNA

Short Interfering RNA

TNFα

Tumor Necrosis Factor alpha

TPM

Transcripts per Million

RPKM

Reads per Kilobase Mapped

TMM

Trimmed Mean or M Values

FDR

False Discovery Rate

ELISA

Enzyme-Linked Immunosorbent Assay

BrdU

Bromodeoxyuridine

cDNA

Complementary DNA

RT-qPCR

Real Time Quantitative PCR

cDNA

Complementary DNA

GAPDH

Glyceraldehyde-3-Phosphate Dehydrogenase

ENCODE

Encyclopedia of DNA Elements

DAB

3,3’-Diaminobenzidine Tetrahydrochloride

MMP9

Matrix Metallopeptidase 9

GEO

Gene Expression Omnibus

FLT3LG

Fms-related Tyrosine Kinase 3 Ligand

SCF

Stem Cell Factor

IL-7

Interleukin 7

IL-15

Interleukin 15

CRISPR

Clustered Regularly Interspaced Short Palindromic Repeats

NLRP3

NLR Family Pyrin Domain Containing 3

Cycle Threshold

Ethics Approval and Consent to Participate

The collection of fetal membrane samples was approved by the Duke Medicine Institutional Review Board with a waiver of consent. As a result, fetal membrane samples were deidentified and there was no link to any clinical information. Fetal membranes samples were collected from term healthy patients at elective cesarean section without prior rupture of membranes or labor using a modification of a previously described protocol.

Consent for Publications

Not applicable.

Availability of data and materials

The gene counts matrix for the RNA-seq libraries are available at the Gene Expression Omnibus repository, under the accession number GSE200312.

All other datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing Interests

The authors declare that they have no competing interests.

Funding

TER, AB, LS, and SJC and supported by grant NHGRI R01 HG010741 and NHGRI R01-HD085227. TKA was supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number KL2TR001115 and the Perinatal Research Fund from the Department of Obstetrics and Gynecology.

The RNA-seq analysis work used a high-performance computing facility partially supported by grants 2016-IDG-1013 (“HARDAC+: Reproducible HPC for Next-generation Genomics") and 2020-IIG-2109 ("HARDAC-M: Enabling memory-intensive computation for genomics") from the North Carolina Biotechnology Center.

Authors’ contributions

SJC made RNA-seq libraries, analyzed gene count matrices, performed all tissue culture for knockdown and subsequent qPCR and ELISAs, performed final overexpression qPCR and ELISA and drafted final manuscript. AB aligned RNA-seq data. LS completed experiments to test overexpression methods. TKA identified patient donors and secured tissue samples and completed all immunohistochemistry staining. TKA and TER participated in interpretation of data and critical revision. All authors were involved in revising the article and gave approval before submission.

Acknowledgement

HEK293T cell line stably expressing dCas9-p300 was gifted from Charlie Gersbach.

Authors Information

Department of Biostatistics and Bioinformatics, Duke University, Durham, NC, USA

SJC, AEB, LS, TER

University Program in Genetics and Genomics, Duke University, Durham, NC, USA

SJC, AEB

Center for Advanced Genomics Technologies, Duke University, Durham, NC, USA

SJC, AEB, LS, TER

Department of Anesthesiology, Duke University Hospital, Durham, NC, USA

TKA

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No competing interests reported.

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Glucocorticoid Receptor Regulated KCNA5 Mediates Cell Proliferation in Human Fetal Membranes

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Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

Background

Methods

Primary Amniotic Epithelial Cell Culture

RNA-seq

RNA-seq read alignment and signal estimation

KCNA5 Knockdown

BrdU ELISA

Active Caspase 3 ELISA

KCNA5 Overexpression

Immunohistochemistry

Results

Glucocorticoid-mediated gene expression responses in the primary amnion epithelial cells

Distribution of KCNA5 expression in human reproductive tissues

Reduced KCNA5 expression increases proliferation in primary amnion epithelial cells but does not affect apoptosis

Recruiting P300 to the KCNA5 promoter increases expression in human embryonic kidney cells

Increased KCNA5 expression reduces proliferation in human embryonic kidney cells

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1