Increased HSD11ß1 Expression in Human Leiomyomatous Uteri: Implication for Enhanced Glucocorticoid Signaling

Abstract: FK506-binding-protein-51 (FKBP51) is a glucocorticoid-induced co-chaperone protein previously shown to bind glucocorticoid receptor (GR), inhibiting its transcriptional activity. We previously found increased FKBP51 levels in uterine leiomyoma versus paired myometrium. To test the hypothesis that elevated FKBP51 levels contribute to leiomyoma pathogenesis by altering GR signaling. Material Method: RNA-sequencing was performed in leiomyoma cell cultures transfected with scrambled or FKBP5-siRNA for 48-h, then treated with vehicle or dexamethasone (DEX) for 24-h. Differentially expressed genes, including HSD11B1, CNN1, and LAMA2 were analyzed by qPCR. Hydroxysteroid 11-beta dehydrogenase 1 (HSD11ß1) expression was analyzed in leiomyoma, leiomyoma-adjacent paired myometrium, myometrium from patients without leiomyoma, and human endometrial stromal cells (HESC) by qPCR and immunohistochemistry in women with or without uterine leiomyoma. HSD11B1 mRNA and protein levels in leiomyoma, paired and normal myometrium, and HESC cells and tissue. Results: HSD11ß1 expression was higher in paired myometrial and leiomyoma tissues versus normal myometrium (P<0.02). DEX treatment increased HSD11B1 transcription in normal myometrial and HESC cultures, but to a significantly greater extent in leiomyoma. However, FKBP5-silencing blunted DEX-induced HSD11B1 transcription. DEX-treatment reduced LAMA2 and increased CNN1 levels (coding for extracellular matrix and smooth muscle proteins, respectively) in FKBP5-silenced versus scrambled-siRNA leiomyoma cultures. Conclusions: FKBP51 not only inhibits but can augment GR-mediated transcription. Importantly, FKBP51-GR interactions increase HSD11B1 levels in leiomyoma cells, generating a pathological FKBP51-GR-HSD11ß1 circle, altering transcription of downstream extracellular matrix and smooth muscle genes to induce a myofibroblast phenotype, thereby possibly contributing to leiomyoma pathogenesis. Overall design: RNA-sequencing was performed in pooled samples from leiomyoma cell cultures transfected with scrambled or FKBP5-siRNA for 48-h, then treated with vehicle or dexamethasone (DEX) for 24-h (n=4/group).

摘要：FK506结合蛋白51（FK506-binding-protein-51，FKBP51）是一种糖皮质激素诱导的辅助伴侣蛋白，既往研究证实其可结合糖皮质激素受体（glucocorticoid receptor, GR）并抑制其转录活性。本团队前期发现，子宫平滑肌瘤组织中FKBP51的表达水平较配对的子宫肌层组织升高。为验证“FKBP51水平升高可通过改变GR信号通路参与平滑肌瘤发病”这一假说，本研究开展相关实验。
材料与方法：将转染了阴性对照siRNA（scrambled siRNA）或FKBP5-siRNA的平滑肌瘤细胞培养48小时后，分别用溶剂对照或地塞米松（dexamethasone, DEX）处理24小时，随后进行RNA测序（RNA-sequencing）。通过实时荧光定量PCR（qPCR）分析差异表达基因，包括HSD11B1、CNN1及LAMA2。采用qPCR与免疫组织化学法，检测伴或不伴子宫平滑肌瘤患者的平滑肌瘤组织、配对癌旁子宫肌层组织、无平滑肌瘤患者的子宫肌层组织以及人子宫内膜基质细胞（human endometrial stromal cells, HESC）中11β-羟类固醇脱氢酶1（Hydroxysteroid 11-beta dehydrogenase 1, HSD11β1）的表达水平，并检测其在平滑肌瘤组织、配对肌层组织、正常肌层组织及HESC细胞与组织中的mRNA与蛋白表达水平。
结果：配对子宫肌层与平滑肌瘤组织中HSD11β1的表达水平均高于正常子宫肌层组织（P<0.02）。地塞米松处理可上调正常子宫肌层细胞与HESC中的HSD11B1转录水平，且该上调效应在平滑肌瘤细胞中更为显著。然而，FKBP5基因沉默可削弱地塞米松诱导的HSD11B1转录上调。在转染FKBP5-siRNA的平滑肌瘤细胞中，地塞米松处理可降低LAMA2的表达水平并上调CNN1的表达（二者分别编码细胞外基质蛋白与平滑肌蛋白）。
结论：FKBP51不仅可抑制GR介导的转录过程，还可增强其转录活性。值得注意的是，FKBP51与GR的相互作用可升高平滑肌瘤细胞中HSD11B1的表达水平，形成病理性的FKBP51-GR-HSD11β1调控环路，通过改变下游细胞外基质与平滑肌基因的转录，诱导肌成纤维细胞表型，进而可能参与平滑肌瘤的发病过程。
整体实验设计：将转染了阴性对照siRNA或FKBP5-siRNA的平滑肌瘤细胞培养48小时后，分别用溶剂对照或地塞米松处理24小时，每组设置4个生物学重复（n=4/group），随后进行RNA测序。