High Mobility Group Box 1 Regulates Uterine Decidualization through Bone Morphogenetic Protein 2 and Plays a Role in Kruppel-Like Factor 5-Induced Stromal Differentiation

Abstract

Background/Aims: High mobility group box 1 (Hmgb1) is associated with a variety of physiological processes including embryonic development, cell proliferation and differentiation, but little information is available regarding its biological role in decidualization. Methods: In situ hybridization, real-time PCR, RNA interference, gene overexpression and MTS assay were used to analyze the spatiotemporal expression of Hmgb1 in mouse uterus during the pre-implantation period, and explore its function and regulatory mechanisms during uterine decidualization. Results: Hmgb1 mRNA was obviously observed in uterine epithelium on day 2 and 3 of pregnancy, but its expression was scarcely detected on day 4 of pregnancy. With the onset of embryo implantation, abundant Hmgb1 expression was noted in the subluminal stromal cells around the implanting blastocyst at implantation sites. Meanwhile, the accumulation of Hmgb1 mRNA was visualized in the decidual cells. Hmgb1 advanced the proliferation of uterine stromal cells and induced the expression of prolactin family 8, subfamily a, member 2 (Prl8a2), a reliable differentiation marker for decidualization. In uterine stromal cells, cAMP analogue 8-Br-cAMP up-regulated the expression of Hmgb1, but the up-regulation was abrogated by protein kinase A (PKA) inhibitor H89. Silencing of Hmgb1 by specific siRNA impeded the induction of 8-Br-cAMP on Prl8a2. Further analysis evidenced that Hmgb1 was a critical mediator of Kruppel-like factor 5 (Klf5) function in stromal differentiation. Knockdown of bone morphogenetic protein 2 (Bmp2) prevented the up-regulation of Prl8a2 elicited by Hmgb1 overexpression, whereas addition of exogenous recombinant Bmp2 protein (rBmp2) reversed the repression of Hmgb1 siRNA on Prl8a2 expression. Conclusion: Hmgb1 may play an important role during mouse uterine decidualization.

High‐mobility group box 1 is a driver of inflammation throughout pregnancy

“…It is thought that this success could be mediated by inflammation‐driven, or coordinated by, HMGB1 within the embryo and uterus (Figure 2). 5‐7,9 …”

“…During embryo development, increased embryonic HMGB1 gene expression promotes the development of the preimplantation embryo by increasing cell number and decreasing apoptosis, therefore enhancing the developmental transition from zygote to blastocyst 5,6 . In addition, increased gene expression of HMGB1 around the implantation site 9 and within the receptive uterus 7 has also been shown to promote pregnancy success, by eliciting a proinflammatory response needed for proper embryo implantation (Figure 2). In addition to an appropriate inflammatory response, the success of pregnancy is widely attributed to progesterone signaling 69 .…”

“…It is thought that this is achieved through the restriction of macrophage accumulation, sustained proinflammatory signaling, and embryo implantation failure 7 . The increased gene expression of HMGB1 within decidual cells has also been shown to induce proliferation and differentiation of uterine stromal cells by up‐regulating the gene expression of bone morphogenetic protein 2 (Bmp2) and stromal cell differentiation marker, cyclic adenosine monophosphate (cAMP) 9 . Collectively, these genes are established as important for successful uterine decidualization 9 .…”

“…Abbreviations: Bone morphogenetic protein 2 (BMP2), cyclic adenosine monophosphate (cAMP), cluster of differentiation (CD14), high-mobility group box 1 protein (HMGB1), interleukin-1 (IL1), interleukin-6 (IL6), interleukin-8 (IL8), lipopolysaccharide (LPS), macrophage inflammatory protein 1 (MCP-1), macrophage inflammatory protein 1 alpha (MIP-1α), myeloid differentiation 2 (MD2), nuclear factor kappa-light chain enhancer of activated B cells (NF-κB), nuclear localization signal 1 (NLS1), nuclear localization signal 2 (NLS2), p38 mitogen-activating kinase (p38), receptor for advanced glycation end products (RAGE), sirtuin 1 (SIRT1), progesterone receptor (PGR), TIR domain-containing adapter-inducing interferon β (TRIF), Toll-like receptor 4 (TLR4), translocating chainassociated membrane protein (TRAM), tumour necrosis factor alpha (TNF-α) mediated by inflammation-driven, or coordinated by, HMGB1 within the embryo and uterus ( Figure 2). [5][6][7]9 Increased HMGB1 expression originates from endometrial and immune cells within the uterus which may influence the proinflammatory signal needed for embryo development and localized inflammatory events at the site of implantation and decidualization. 5,7,9 During embryo development, increased embryonic HMGB1 gene expression promotes the development of the preimplantation embryo by increasing cell number and decreasing apoptosis, therefore enhancing the developmental transition from…”

“…However, the exact mechanism(s) underpinning how HMGB1 augments this inflammation is still unclear. HMGB1 also plays an important role in uterine decidualization by mediating the expression of key stromal cell markers that induce the proliferation and differentiation of uterine stromal cells 9 . Although the inflammatory response mediated by HMGB1 in the early stages is critical for pregnancy success, excessive HMGB1 levels within the uterine cavity 8 and expression within the placenta 10,11 have been shown to lead to pregnancy loss.…”