• Keratinocyte growth factor and vascular endothelial growth factor: Mediators of progesterone action in the uterus and ovary

      Rocca, Meredith Schneider; Koos, Robert D. (1999)
      Progesterone plays an important role in mammalian reproduction, but little is known about mediators of its action. In this dissertation I investigated two growth factors that may mediate progesterone's action in two critical target tissues - keratinocyte growth factor (KGF) in the mouse uterus and vascular endothelial growth factor (VEGF) in the rat ovary. Under the influence of progesterone, the mouse uterine epithelium undergoes dramatic remodeling during the estrous cycle and pregnancy. KGF-KGFR signaling plays a role in mesenchymal-epithelial cell communication in a variety of tissues. I examined the expression of KGFR, and its known ligands in the mouse uterus by RT-PCR. I found that KGFR, KGF, FGF-1, and FGF-10, but not FGF-3 are expressed. Uterine mRNA levels for KGFR and aFGF did not vary with hormone treatment. However, KGF mRNA expression increased coincident with the preovulatory progesterone surge and decreased with estrogen treatment. In contrast, FGF-10 expression increased in the post-ovulatory period and was unaffected by estrogen treatment. These findings suggest that KGF and FGF-10 play a role in steroid-induced remodeling of the mouse uterus. To study the role of KGFR signaling, a transgenic mouse was produced with a dominant-negative mutant KGFR targeted to be expressed in the uterus. Expression of the transgene could not be demonstrated in the uterus, however, and no conclusions can be drawn from our experiments on the role of uterine KGFR signaling. In the rat, a preovulatory surge of progesterone is essential for ovulation. Just prior to ovulation, the follicle swells with edema, monocytes infiltrate, and the follicle wall is broken down by plasmin and collagenases. VEGF expression has also been observed to increase subsequent to the preovulatory progesterone surge and may mediate these ovulatory events. I examined the expression of VEGF in the rat ovary by RT-PCR. Inhibition of the preovulatory progesterone surge inhibited both ovulation and VEGF mRNA expression in the rat. Progesterone appears to act via its receptor to induce VEGF mRNA expression, as treatment with a progesterone receptor antagonist also suppressed VEGF expression. The correlation of VEGF expression and ovulation suggests that VEGF plays a role in ovulation in the rat.
    • Steroid control of growth factor gene expression in the rat uterus: The role of vascular endothelial growth/permeability factor in the regulation of estrogen-induced increases in uterine vascular permeability and growth

      Cullinan-Bove, Kathleen; Koos, Robert D. (1995)
      In the uterus, estrogen from the ovary stimulates microvascular permeability and edema, followed by proliferation of both epithelial cells and cells of the richly vascular stroma. These responses can be mimicked in an ovariectomized (ovx) animal by administering 17{dollar}\beta{dollar}-estradiol (E{dollar}\sb2{dollar}). Uterine hypertrophy and hyperplasia may be the result of an E{dollar}\sb2{dollar}-induced increase in locally produced growth factors. This dissertation investigates the expression of several growth factors in the uterus by E{dollar}\sb2{dollar}, and correlates expression patterns of these growth factors with the E{dollar}\sb2{dollar}-induced uterotropic responses. Using the specific and sensitive method of reverse transcription-polymerase chain reaction (RT-PCR), we have shown that E{dollar}\sb2{dollar} administered to immature, ovx animals regulates the expression of several growth factors in a time dependent manner. mRNA for acidic fibroblast growth factor (aFGF), basic FGF (bFGF), keratinocyte GF (KGF) and transforming GF-beta{dollar}\sb2{dollar} (TGF-{dollar}\beta\sb2{dollar}) are all expressed in the rat uterus and their expression is enhanced by E{dollar}\sb2{dollar} treatment (6 h). Also, E{dollar}\sb2{dollar} regulation of mRNA levels for vascular endothelial growth/permeability factor (VEG/PF) was investigated. As early as 30 minutes after E{dollar}\sb2{dollar} treatment, mRNA levels for VEG/PF begin to increase and reach maximal levels at 2 h (8-fold higher than control levels). Preliminary studies show that following E{dollar}\sb2{dollar}-treatment, VEG/PF mRNA levels are highest in uterine epithelial cells. Metabolic labelling and immunoprecipitation have shown that E{dollar}\sb2{dollar} stimulates a corresponding increase in uterine VEG/PF protein production. The increase in VEG/PF levels immediately precedes the known increase in E{dollar}\sb2{dollar}-induced uterine water imbibition, and suggests that VEG/PF, a potent stimulator of vessel permeability, might play a role in this uterotropic response. The interaction of E{dollar}\sb2{dollar} and progesterone (P{dollar}\sb4{dollar}) on the regulation of VEG/PF and bFGF expression in the immature, ovx animal was also investigated. Initial studies showed that treatment of immature, ovx rats with P{dollar}\sb4{dollar} alone stimulated the expression of VEG/PF similar to the effect of E{dollar}\sb2{dollar}. However, when P{dollar}\sb4{dollar} and E{dollar}\sb2{dollar} were administered together, after 48 h of E{dollar}\sb2{dollar}-priming, P{dollar}\sb4{dollar} suppressed the E{dollar}\sb2{dollar}-induced increase in VEG/PF mRNA but had no effect on the E{dollar}\sb2{dollar}-induced increase in bFGF mRNA. Finally, the ability of recombinant human VEG/PF to mimic the effect of E{dollar}\sb2{dollar} in increasing uterine vessel permeability by direct injection into the uterine lumen was investigated. Similarly we attempted to inhibit the effects of E{dollar}\sb2{dollar} by treating the animals, intraluminally, with antisense VEG/PF and anti-VEG/PF antiserum. These studies were not successful. The E{dollar}\sb2{dollar}-induced uterine proliferative response most likely involves a complex interaction of a variety of growth factors. The data have shown that E{dollar}\sb2{dollar} stimulates expression of several growth factors in the rat uterus. The rapid E{dollar}\sb2{dollar}-induced increase in VEG/PF mRNA (2 h), suggests that VEG/PF expression may be a prerequisite for the subsequent expression or action of other growth factors present in the uterus.
    • The uterotrophic effects of relaxin: Activation of ligand-independent estrogen receptor-mediated transcription

      Pillai, Suresh Biswanath; Koos, Robert D. (2001)
      The peptide hormone/growth factor relaxin has numerous and diverse effects in the cardiovascular system, the nervous system, and throughout the female reproductive tract. In females, relaxin is involved with the processes of cervical ripening and relaxation of the pubic symphysis prior to parturition, mammary gland and nipple development, and uterine growth and metabolism. The time course and nature of relaxin's effects on the uterus closely mirror those of the steroid hormone estradiol (E2), suggesting that the signaling pathways of relaxin and E2 may share some common components. This dissertation examines the relationship between relaxin and E2-mediated signaling in the uterus and in breast cancer cells. In immature, ovariectornized rats, relaxin and E2 both induced increases (75% and 100%, respectively) in the uterine wet weight/body weight ratio versus controls 6 hours following treatment. These increases were completely inhibited by pre-treatment with ICI 182 780, a potent and specific estrogen receptor (ER) antagonist. Histological analysis of uterine cross sections revealed that the uterine edema was localized to the endometrium; ICI 182 780 pre-treatment blocked this edema. From these data, we concluded that relaxin's uterotrophic effects involved ligand-independent activation of ERs. RT/PCR examination of uterine mRNA from ovariectomized rats treated with relaxin or E2 showed that both significantly and rapidly up-regulated vascular endothelial growth factor (VEGF) and progesterone receptor expression. These experiments were followed by RT/PCR analysis of ERbeta regulation, which revealed that both relaxin and E2 down-regulated uterine ERbeta1 and ERbeta2 in vivo. This is the first report of E2 regulation of ERbeta2 and of relaxin regulation of ERbeta regulation. To confirm that relaxin activates ERs and ER-mediated transcription, we transfected relaxin and E2 responsive MCF-7 cells with an ERE-Luc reporter gene vector and treated them with relaxin and E2. Luciferase expression increased significantly in response to both relaxin and E 2; these increases were blocked by co-treatment with ICI 182 780. From these data, we concluded that relaxin increased reporter gene expression through ligand-independent stimulation of ER-ERE-mediated transcription. In summary, relaxin's trophic effects on the uterus involve, to a large part, activation of ERs.