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Dual SMAD inhibition enhances the longevity of human epididymis epithelial cells.

ABSTRACT: Primary human epididymis epithelial (HEE) cells are valuable reagents for functional studies on the human epididymis. We used them previously to determine the transcriptional networks that establish cell identity along the length of the epididymis from caput, corpus, and cauda. These studies on HEE cells and organoids derived from them revealed important cellular properties. However, similar to other primary cells, HEE cells undergo replicative senescence and de-differentiation in culture. A cocktail of small molecules was shown elsewhere to extend longevity of epithelial basal cells. The components included transforming growth factor β (TGF-β)/bone morphogenetic protein (BMP) antagonists, WNT agonist, and Rho-associated and coiled-coil containing protein kinase (ROCK) inhibitor (ROCKi), which together prevented the senescence-related upregulation of TGF-β signaling pathway members. Here, we treat HEE cells with the same cocktail and observed enhanced replicative potential and prolonged expression of markers of HEE differentiation. This treatment expands the differentiated HEE cell population available from individual epididymis tissue samples that can be used for molecular, cellular, and functional studies.

SUBMITTER: Coatti GC

PROVIDER: S-EPMC9713186 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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Dual SMAD inhibition enhances the longevity of human epididymis epithelial cells.

Coatti Giuliana C GC Paranjapye Alekh A Harris Ann A

Cell and tissue research 20221201 2

Primary human epididymis epithelial (HEE) cells are valuable reagents for functional studies on the human epididymis. We used them previously to determine the transcriptional networks that establish cell identity along the length of the epididymis from caput, corpus, and cauda. These studies on HEE cells and organoids derived from them revealed important cellular properties. However, similar to other primary cells, HEE cells undergo replicative senescence and de-differentiation in culture. A coc ...[more]

PMID: 36454271

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Project description:Study questionWhat is the transcriptional network governed by the androgen receptor (AR) in human epididymis epithelial (HEE) cells from the caput region and if the network is tissue-specific, how is this achieved?Summary answerAbout 200 genes are differentially expressed in the caput HEE cells after AR activation; the AR transcriptional network is tissue-specific and may be mediated in part by distinct AR co-factors including CAAT-enhancer binding protein beta (CEBPB) and runt-related transcription factor 1 (RUNX1).What is known alreadyLittle is known about the AR transcriptional program genome wide in HEE cells, nor its co-factors in those cells. AR has been best studied in the prostate gland epithelium and prostate cancer cell lines, due to the important role of this factor in prostate cancer. However AR-associated differentially expressed genes (DEGs) and AR co-factors have not yet been compared between human epididymis and prostate epithelial cells.Study design, size, durationCaput HEE cells from two donors were exposed to the synthetic androgen R1881 at 1 nM for 12-16 h after 72 h of hormone starvation.Participants/materials, setting, methodsChromatin was prepared from R1881-treated and vehicle control HEE cells. AR-associated chromatin was purified by chromatin immunoprecipitation (ChIP) and AR occupancy genome wide was revealed by deep sequencing (ChIP-seq). Two independent biological replicates were performed. Total RNA was prepared from R1881 and control-treated HEE cells and gene expression profiles were documented by RNA-seq. The interaction of the potential novel AR co-factors CEBPB and RUNX1, identified through in-silico motif analysis of AR ChIP-seq data, was examined by ChIP-qPCR after siRNA-mediated depletion of each co-factor individually or simultaneously.Main results and the role of chanceThe results identify about 200 genes that are differentially expressed (DEGs) in HEE cells after AR activation. Some of these DEGs show occupancy of AR at their promoters or cis-regulatory elements suggesting direct regulation. However, there is little overlap in AR-associated DEGs between HEE and prostate epithelial cells. Inspection of over-represented motifs in AR ChIP-seq peaks identified CEBPB and RUNX1 as potential co-factors, with no evidence for FOXA1, which is an important co-factor in the prostate epithelium. CEBPB and RUNX1 ChIP-seq in HEE cells showed that both these factors often occupied AR-binding sites, though rarely simultaneously. Further analysis at a single AR-regulated locus (FK506-binding protein 5, FKPB5) suggests that CEBPB may be a co-activator. These data suggest a novel AR transcriptional network governs differentiated functions of the human epididymis epithelium.Large scale dataAR ChIP-seq and RNA-seq data are deposited at GEO: GSE109063.Limitations, reasons for cautionThere is substantial donor-to-donor variation in primary HEE cells cultures. We applied stringent statistical tests with a false discovery rate (FDR) of 0.1% for ChIP-seq and standard pipelines for RNA-seq so it is possible that we have missed some AR-regulated genes that are important in caput epididymis function.Wider implications of the findingsOur data suggest that a novel AR transcriptional network governs differentiated functions of the human epididymis epithelium. Since this cell layer has a critical role in normal sperm maturation, the results are of broader significance in understanding the mechanisms underlying the maintenance of fertility in men.Study funding/competing interestsThis work was funded by the National Institutes of Health, Eunice Kennedy Shriver National Institute of Child Health and Development: R01 HD068901 (PI: Harris). The authors have no competing interests to declare.

Dataset Information

Dual SMAD inhibition enhances the longevity of human epididymis epithelial cells.

Publications

Dual SMAD inhibition enhances the longevity of human epididymis epithelial cells.

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