Mechanisms of the Progestational Regulation of Tissue Factor and Plasminogen Activator Inhibitor in the Endometrium

Graciela Krikun, Ph.D.

In humans, progesterone stimulation of the estradiol-primed endometrium initiates decidualization of the stromal cells around the spiral arterioles to establish the decidual cell as a major cell type of the secretory phase and pregnant endometrium. Prior studies from our laboratory have established that progestins stimulate the endometrial decidual/ stromal expression of two crucial modulators of hemostasis: tissue factor (TF) and plasminogen activator inhibitor type 1 (PAI-1). Neither one of these genes contains consensus estrogen or progesterone response elements My studies focused on the mechanisms underlying the enhanced expression by progestin of TF and PAI-1 in vivo as well as in primary human endometrial stromal cell cultures. It was determined that the prolonged progestational upregulation occurred at the transcriptional level. Transient transfections of human endometrial stromal cells were carried out with truncated TF or PAI-1 promoters as well as promoters undergoing site directed mutagenesis. These studies identified specific Sp1 sites as the key regulators of progestin-induced activity of the TF and PAI-1 genes. Co-expression of these promoters with overexpression vectors for Sp1 and/or Sp3 demonstrated the crucial role for Sp1 but not Sp3 in the transcriptional regulation of both TF and PAI-1 (see figure below). Most interestingly, immunohistochemical studies demonstrated increased Sp1 levels in perivascular stromal cells in secretory phase compared to proliferative phase endometria. Conversely, Sp3 expression was greatly decreased in stromal cells of secretory compared to proliferative phase tissues. We proposed that the changes observed in the cycling endometrium altered the ratio of Sp1 to Sp3 transcription factors and that these changes in turn were involved in TF and PAI-1 gene expression in vivo and in vitro.

Long Term Progestin Only Contraception and Abnormal Uterine Bleeding.
Recent studies have focused on the effects of long term progestin only contraception (LTPOC) on aberrant angiogenesis. Although LTPOCs are highly effective and safe, irregular and prolonged endometrial breakthrough bleeding is the primary reason for discontinuing their use. Histological sections of endometria from LTPOC-treated patients display abnormally enlarged blood vessels at bleeding sites. Paradoxically, a trend towards reduced endometrial perfusion in LTPOC users has been reported in these patients. We hypothesized that hypoxia/re-perfusion-induced free radical production inhibits the expression of angiopoietin-1 (Ang-1), a vessel stabilizing factor, leaving unopposed the effects of endothelial Ang-2, a vessel branching and permeability factor. Immunohistochemical studies confirmed selective decreases in stromal cell Ang-1 in LTPOC-exposed endometrium. To indirectly assess whether LTPOC enhances endometrial free radical production, immunostaining was conducted for the phosphorylated form of the stress-activated kinases SAPK/JNK and p38. These kinases were greatly increased in endometria from LTPOC-treated patients. Hence, we are examining the effects of progestin, hypoxia and reactive oxygen species on the regulation of Ang-1 and 2 as well as the activation of MAPK, SAPK/JNK and p38 by cultured human endometrial stromal cells and human endometrial endothelial cells. The effect of these treatments on the production of lipid peroxides will also be conducted.

Understanding the mechanism of LTPOC induced endometrial bleeding is expected to lead to therapies that will alleviate or prevent this undesirable side effect.