The androgen receptor mediates antiapoptotic function in myometrial cells

Abstract

During pregnancy, myometrial phenotype is programmed into three characteristic stages referred to as the early proliferative, the midterm hypertrophic, and the late contractile stage. Increased myometrial growth in the early and midterm of pregnancy involves a complex process of cell proliferation, antiapoptosis and differentiation. We have previously demonstrated that the androgen receptor (AR) is required for myometrial cell proliferation by modulating IGF-1 signaling during early pregnancy. Here, we report that AR also exerts its antiapoptotic function in human myometrial cells. Enhanced AR expression protects, whereas AR silencing sensitizes myometrial cells to both intrinsic and extrinsic apoptotic stimuli. AR agonist inhibits, whereas AR antagonist induces myometrial cells to undergo apoptotic cell death. Gene microarray analysis confirms that the central functions of AR in myometrial cells are to regulate cell cycling and apoptosis through three major gene groups involving the epidermal growth factor (EGF) signaling, RNA splicing and DNA repair processes. AR mediates its antiapoptotic function through two distinct pathways. In the receptor-dependent pathway, AR is required for the expression of several protein factors within the EGF signaling pathway. Through the PI3K/Akt pathway, AR enhances the expression of the antiapoptotic protein Mcl-1. In the ligand-dependent pathway, AR agonist triggers the activation of Src kinase, which in turn phosphorylates STAT3 to increase Mcl-1 expression. We conclude from these results that the AR signaling exerts antiapoptotic function in myometrial cells, further supporting its key role in programming of myometrial phenotype.

Main

The myometrium demonstrates remarkable plasticity during pregnancy, manifest by changes in myometrial phenotype across pregnancy starting from the early proliferative, to the midterm synthetic and the later contractile stages.^{1, 2} It concludes with postpartum uterine remodeling to complete the reproductive cycle following labor and returns to its nonpregnant receptive state. The proliferative stage of pregnancy involves a complex process of cell proliferation, apoptosis as well as differentiation. At this stage, the myometrial phenotype is characterized by an enhanced proliferation index for example, PCNA and BrdU incorporation.³ In addition, emerging evidence suggests that antiapoptotic mechanisms are also developed to cooperate with myometrial growth. Using a pregnant rat model, we showed high levels of the antiapoptotic proteins, Bcl2 and Bcl2L1, expressed throughout the proliferative stage.³ In addition, although there are several executioner caspases (e.g. caspase 3, 6 and 7) activated during this stage, apoptotic cell death is not observed in myometrial cells.^{3, 4, 5} As the early proliferative phase of pregnancy is essential to accumulate enough myometrial cell numbers and contractile capacity to engage in parturition, these observations suggest that antiapoptotic mechanisms must be coordinately developed along with cell proliferation during early pregnancy to achieve optimal myometrial growth. Thus, investigation of the antiapoptotic mechanisms of myometrial cells will contribute significantly to our understanding the physiology of uterus and the clinical management of pregnancy and labor progression.

Signaling mediated by estrogen and progesterone receptors had been demonstrated to be important for myometrial growth.^{6, 7, 8, 9} As transcriptional factors, these steroid receptors regulate expression of several growth factors to modulate myometrial cell growth.^{10, 11, 12} Growth factors such as insulin-like growth factor (IGF) and epidermal growth factor (EGF) bind their cognate tyrosine kinase receptors and trigger multiple downstream signaling cascades such as PI3K/Akt and Ras-Erk/MAPK.^{13, 14} Steroid receptors can also exert non-genomic actions to control cell proliferation. Ligand-activated receptors can recruit Src kinase and activate its downstream signaling cascade.^{15, 16} Therefore, the mutual interplay between steroid receptors and growth factors are vital for myometrial growth.

The androgen receptor (AR), a nuclear steroid receptor, is also expressed in the myometrium. Serum androgen levels start to increase progressively during the pregnant luteal phase, initiated by the luteinizing hormone surge.¹⁷ AR expression levels in the myometrium are also maintained at higher levels during early proliferative and synthetic phases of pregnancy.^{Supplementary Table.}

UV light and anti-Fas antibody induced apoptosis

cells were serum starved for 12 h before exposured to UV light (UV Stratalinker 1800) for various doses. Cells were allowed to recover for the indicated periods of time. To induce apoptosis through the extrinsic pathway, cells were treated with anti-Fas antibody (clone CH110) at various doses and time points.²⁰ Cells were harvested and the percentage of cells with Annexin V binding was determined by flow cytometry.²⁰ Protein lysates was extracted for western blotting of c-PARP1 and c-CASP3.

Flow cytometry

Annexin V-PE/7-AAD Apoptosis Detection Kit (BD Pharmingen, San Diego, CA, USA) were used to detect apoptotic cell populations according to manufacturer’s protocol. Briefly, the cells (1 × 10⁵ cells) were suspended in 100 μl binding buffer, and then treated with 5 μl of Annexin V-PE (BD Pharmingen) and 5 μl 7-AAD before analyzed on FACSCanto II flow cytometer and BD FACSDiva software v5.0.3 (Becton Dickinson, Franklin, NJ, USA). Ten thousand events are acquired for statistical analysis. Detection of apoptotic population was performed according to the manufacturer’s instructions (https://www.bdbiosciences.com/external_files/pm/doc/tds/cell_bio/live/web_enabled/6900KK_559763.pdf).

Microarray and Real-time PCR

Gene microarray analyses were performed as described before.⁴⁰ Briefly, Total RNA was extracted from hTERT(shAR) and hTERT(shCTRL) cells by the mirVana RNA Isolation Kit (Ambion, Austin, TX, USA) from three independently repeated experiments. The quality and quantity of RNA were assessed with an Agilent 2100 Bioanalyzer (Caliper Technologies Corp., CA, USA). Amplified and Alexa Fluor 3 labeled RNA samples from hTERT(shCTRL) and hTERT(shAR) cells were hybridized onto the Human Agilent 4 × 44k (Agilent Technologies, Santa Clara, CA, USA), along with Alexa Fluor 5 labeled human reference RNA. Hybridization signals were analyzed following the manufacturer’s instruction. Statistical Analysis of Microarray (SAM) program (http://www-stat.stanford.edu/tibs/SAM/) was used to analyze expression differences between RNA samples from hTERT(shAR) and hTERT(shCTRL) cells. Unpaired t-tests were calculated for all probes passing filters and controlled for multiple testing by estimation of q-values using the false discovery rate method.⁴¹ IPA software (Ingenuity Systems) was used to analyze AR-regulated gene groups and signaling pathways.

Real-time PCR assays

Total RNA was extracted using Purelink RNA mini kit (Invitrogen, Burlington, ON, Canada) according to the manufacturer’s instructions. Two micrograms of total RNA was subjected to a random-primed reverse transcription using M-MLV reverse transcriptase (Invitrogen). Real-time qPCR was conducted in triplicates using Applied Biosystems7900HT with 5 ng of cDNA, 1 μM of each primer pair and SYBR Green PCR master mix (Roche, Mississauga, ON, Canada). The sequences of primers were shown below. Relative mRNA levels were normalized to GAPDH.

Statistics

Results are expressed as the mean±S.E.M. To determine differences between two groups, student t-test was carried out using GraphPad Prism (version 4) with the level of significance set at P<0.05 as *, P<0.01 as ** and P<0.001 as ***.