This study aimed to characterise the expression and regulation of class IIa HDACs in cyclic human endometrium and endometrial stromal cells, in vitro. Our results imply a cyclic expression pattern of Class IIa HDACs in human endometrial tissues, with upregulation during the early secretory and mid secretory phase. Furthermore, hormonal treatment of stromal cells showed significant changes in protein expression levels of class IIa HDACs in response to different combinations of steroid hormone treatments, with an increase in expression observed after treatment with combined estrogen and progesterone for 24 hrs in HDAC7 and 48 hrs in HDACs 4 and 9a. The treatment of HESCs with TSA combined with steroid hormones also demonstrated a significant inhibitory effect on HDACs 5, 7, and 9 expressions.
Post menstruation, the endometrial tissue lining starts to regenerate and proliferate, preparing for the next cycle. Increasing evidence suggests that Class 1 HDACs play a role in cell proliferation, and overexpression of HDACs 1, 2, and 3 proteins in cells have been implicated in endometrial cancer [28-31]. There are no studies on class IIa HDACs in the endometrium. However, other studies on class IIa HDACs suggest that they may be involved in tissue-specific development and differentiation [32-34]. HDAC5 is seen to be downregulated in several cancers, including prostate and breast cancer. CDK4/6 inhibitors inhibit CDK functions and thus induce cell cycle arrest and are being studied as an endometrial cancer treatment . It is observed that HDAC5 deficient cells are linked to CDK4/6 inhibitor resistance in breast cancer cells , leading to cell proliferation. Our endometrial tissue mRNA expression data shows class IIa HDAC 5 downregulated during the proliferative stage compared to the early secretory phase suggesting its possible negative regulation of proliferation.
In the endometrium, the secretory phase is marked by decidualization, which is the differentiation of human endometrial stromal cells in preparation of trophoblast invasion and subsequently implantation [37, 38]. It is a complex mechanism regulated by several factors . An increase in H3K27ac is observed in HESCs during Decidualization, acting as an enhancer of insulin-like growth factor binding protein-1 (IGFBP-1), a decidualization marker . In addition, the decidual-like morphology is induced by the action of progesterone on estrogen primed endometrium in combination with cyclic adenosine monophosphate (cAMP) [2, 41, 42]. Previous studies have reported the involvement of class IIa HDACs in cAMP signaling in various cell types , suggesting that a similar correlation could be involved in endometrial cells as well.
TSA has been observed to inhibit HDACs and enhance decidualization . A study on endometrial stromal cells showed that TSA inhibits HDACs and subsequently enhances expression of decidualization markers insulin-like growth factor binding protein-1 (IGFBP-1) and prolactin . TSA-treated HESCs inhibit HDAC and promote histone acetylation at the promoters of TIMP-1 and TIMP-3 by increasing the transcription of these genes . Alternatively, another study showed that inhibition of HDAC5 by TSA decreases cAMP-induced expression of inducible nitric oxide synthase (iNOS) during HESC decidualization, demonstrating a role for HDAC5 in decidualization. We observed an overall downregulation in class IIa HDACs 5, 7, 9a, and 9b protein expression in HESCs treated with combined TSA and steroid hormones. Thus, demonstrating that TSA mitigates the effect of steroid hormones in HESCs
During the late secretory phase, falling progesterone levels trigger an inflammatory response, marked by chemo- and cytokines production by decidualized stromal cells [37, 47, 48]. Class IIa HDACs, especially HDAC 4, have been shown to induce inflammatory responses [49-51]. Class IIa HDACs regulate cytokine expressionn, such as HDAC4 regulates IL-10 expression , while HDAC5 binds to IL-8 promoter resulting in inhibition of IL-8 expression [51, 53]. IL-2 expression is inhibited by HDAC7 and 9 interaction with FOXP3 and TIP60 [51, 54]. Class IIa HDACs 4 and 7 have been involved in regulating a crucial pro-inflammatory transcription factor Hypoxia-inducible factor (HIF)-1α, which is essential for vascularization in the premenstrual phase [50, 55, 56]. Significant downregulation was observed in HDACs 5, 7, and 9 during the late secretory phase in the endometrial cycle, while there was no significant change in HDAC4 expression throughout. Class IIa HDACs could be involved in inflammatory responses in the endometrium.
Our data suggest that the class IIa HDACs might be involved more prominently in regulating gene expression during the secretory phase due to their upregulation during the early and mid-secretory phases. Similarly, in steroid hormone-treated HESCs, a significant increase in class IIa HDACs was observed upon the addition of progesterone which is known to induce decidualization in HESCs [41, 42]. Decidual HESCs make the significant component of the human decidua . This study focused on the HESC cell line to observe class IIa importance in secretory changes. Besides stromal cells, human decidua also comprises glandular cells, immune cells, and blood and lymph vessels . Further studies on class IIa expression in immune cells and epithelial cell lines would better help us understand their menstrual cycle function. The mRNA expression levels of class IIa HDACs in HESCs were too low to be analyzed, but significant protein expression was observed, which could be due to rapid mRNA turnover [58, 59].
Two bands were observed for HDAC9 (9a and 9b). A similar presence of two isoforms for HDAC9 has been observed previously in other tissues and cells [60, 61]. HDAC9 has multiple alternatively spliced isoforms. In breast cancer cells, two isoforms have been observed one is a full-length HDAC9 with low expression levels and a truncated version that lacks the c-terminal deacetylase domain and is highly expressed in breast cancer cells . Understanding the structural and functional differences in the two observed isoforms of HDAC9 in endometrial cells will give us a better understanding of their function in the endometrium. Further investigation is required to verify if there are any similarities between isoforms observed in this study and found in other tissues.
Class IIa HDACs have the unique ability to shuttle between nucleus and cytoplasm, resulting from post-translational modifications (PTMs) . Other than regulation of subcellular localization, class IIa activities are also regulated by other PTMs, such as ubiquitin-dependent degradation . In addition, they function as complexes by associating with other HDACs such as HDAC3 . There is strong evidence of crosstalk between HDACs and other epigenetic factors in regulating cancer tumorigenesis [22, 54, 64].
Histone acetylation and HDACs have been involved in endometrial pathologies such as endometrial carcinomas and endometriosis [65, 66]. This study suggests the involvement of class IIa HDACs in the menstrual cycle regulation, most prominently in the differentiation of endometrium.