Activins regulate FSH synthesis by stimulating the phosphorylation and nuclear accumulation of SMAD2 and SMAD3, which bind to a consensus SMAD-binding element in the proximal murine FSHβ (Fshb) subunit gene to drive transcription. Previous over-expression and in vitro DNA binding analyses suggested that SMAD4 participates in complexes with SMAD2 and SMAD3 to regulate Fshb expression. Here, we have characterized the role of endogenous SMAD4 in activin A induction of Fshb transcription in immortalized murine gonadotropes (LβT2). We identified five murine Smad4 mRNA isoforms, of which, four are newly described; however, the canonical full-length form predominated at both the mRNA and protein levels. Depletion of endogenous SMAD4 by RNA interference (RNAi) abolished activin A-induced Fshb promoter-reporter activity and greatly attenuated constitutively active activin type IB receptor-stimulated Fshb mRNA levels. The activin A response was rescued with an RNAi-resistant form of wild-type SMAD4, but not with a DNA-binding-deficient (Lys88Arg) SMAD4, suggesting that DNA binding by SMAD4 is necessary for activin induction of the Fshb gene. Though SMAD2 and SMAD3 are generally thought to partner with SMAD4 prior to accumulation in the nucleus, treatment with leptomycin B, an inhibitor of SMAD4 nuclear export, reduced but did not prevent activin A induction of Fshb mRNA levels or promoter activity. In addition, a constitutively nuclear form of SMAD4 rescued the effect of endogenous SMAD4 depletion. Collectively, these data demonstrate a necessary role for SMAD4 in activin A induction of the murine Fshb gene in immortalized gonadotropes.
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Ying Wang, Vanessa Libasci, and Daniel J Bernard
F Wang, R Duan, J Chirgwin, and SH Safe
Insulin-like growth factor-I (IGF-I), transforming growth factor alpha (TGFalpha) and epidermal growth factor (EGF) induced cathepsin D gene expression and reporter gene activity in MCF-7 human breast cancer cells transiently transfected with a construct (pCD1) containing a -2576 to -124 cathepsin D gene promoter insert. In contrast, IGF-I, but not TGFalpha or EGF, induced reporter gene activity in cells cotransfected with wild-type estrogen receptor (ER) expression plasmid and a construct (pCD2) containing estrogen-responsive downstream elements from -208 to -101. Promoter deletion and mutational analysis experiments identified four GC-rich sites and an imperfect palindromic estrogen responsive element required for IGF-I activation of the ER (ligand-independent). Subsequent studies with the mitogen-activated protein kinase (MAPK) inhibitor, PD98059, and a serine(118(-ER mutant confirmed the role of the MAPK pathway for IGF-I activation of the ER in MCF-7 cells. Thus, growth factor activation of ER can mediate transactivation vs ER/Sp1 binding to GC-rich sites and represents a novel pathway for ligand-independent ER action. The divergent pathways for IGF-I and TGFalpha/EGF activation of the ER observed in MCF-7 cells contrast with previous data indicating that pathways for growth factor activation of the ER are dependent on the gene and/or gene promoter and on cell context.
Y.-J. Y. Wan, L. Wang, and T.-C. J. Wu
The presence of retinoic acid receptor (RAR) α, β and γ mRNA was examined in 16 different kinds of rat tissue using the highly sensitive reverse transcriptase-polymerase chain reaction technique. The data demonstrated that each tissue expressed at least two types of RAR mRNA. Among the three types of RAR mRNA, RAR α was widely expressed in all types of organ and was the dominant form expressed in the gastrointestinal tract. RAR β mRNA was not present in the intestine and spleen. In addition, RAR β mRNA levels were high in the heart, lung, brain, testis and epididymis. RAR γ mRNA was abundant in both male and female reproductive systems, as well as epidermal tissues. The prevalence of each RAR mRNA in the tissues suggests the diverse biological roles of these receptors.
Y-J Y Wan, L Wang, and T-C J Wu
Mouse embryonal carcinoma F9 cells are pluripotent stem cells and differentiate into primitive endodermal cells upon treatment with retinoic acid (RA). We have recently shown that in F9 cells RA regulates gene expression of activin receptor type II (ActR-II), whose ligand is a potent differentiation agent. The present study examined the regulation of the newly cloned activin receptor type IIB (ActR-IIB) gene by RA. F9 cells expressed equal amounts of three ActR-IIB transcripts of 8·0, 7·5 and 4·0 kb. Both 9-cis-RA (c-RA) and all-trans-RA (t-RA) induced ActR-IIB gene expression in a dose-dependent manner. At 10−9 m c-RA exerted no effect, while 10−5 m c-RA increased the 8·0 kb ActR-IIB transcript about sevenfold. In contrast, t-RA induced the 8·0kb ActR-IIB transcript fivefold at 10−9 m and up to eightfold at 10−5 m. The inductive effect on the 8·0 kb transcript was greater than that on the 7·5 kb transcript, and was least effective on the 4·0 kb transcript, suggesting that these three mRNA isoforms may originate from different promoters. Both cycloheximide and actinomycin D inhibited the inductive effect of t-RA on ActR-IIB gene expression, in contrast to ActR-II whose gene expression was not suppressed by cycloheximide but abolished by actinomycin D. Thus, endodermal differentiation of F9 cells is associated with activation of ActR-IIB gene and the mechanisms involved in the regulation of ActR-II and IIB gene expression are different.
Y-J Y Wan, T Pan, L Wang, J Locker, and T-C J Wu
In McA-RH 8994 rat hepatoma cells, all-transretinoic acid (t-RA) induces expression of the α-fetoprotein (AFP) and albumin genes and results in a phenotype similar to differentiated fetal hepatocytes. The present study elucidated the mechanism involved in AFP gene regulation mediated by retinoic acid. Northern blot analyses demonstrated that 9-cis-retinoic acid (c-RA), a ligand for retinoid x receptors (RXRs), also induced expression of the AFP gene in McA-RH 8994 cells. The induction was time- and dose-dependent. Northern blots and transfection assays using the 7·3 kb full-length regulatory region of the AFP gene demonstrated that c-RA was more effective than t-RA in regulating expression of the AFP gene. At 10−7 m, c-RA increased AFP mRNA 5-fold and chloramphenicol acetyltransferase (CAT) activity 2·5-fold. In contrast, t-RA at a concentration of 10−7 m exerted no significant effect; 10− 6 to 10−5 m t-RA was needed to affect AFP gene expression. These data suggested that activation of RXRs is essential for the regulation of the AFP gene. Co-transfection experiments revealed that overexpression of RXRα in McA-RH 8994 cells further enhanced the CAT activity induced by c-RA. In addition, c-RA did not alter the half-life of AFP mRNA. Thus, RXRα may play a crucial role in transcriptional regulation of the AFP gene and in controlling hepatocyte phenotype.
I Chakraborty, S K Das, J Wang, and S K Dey
Cyclo-oxygenase (COX) is a rate-limiting enzyme that converts arachidonic acid to prostaglandins (PGs) and exists in two isoforms, COX-1 and COX-2. In the rodent, increased uterine vascular permeability at sites of blastocyst apposition is one of the earliest prerequisite events in the implantation process. This event is preceded by generalized uterine edema and luminal closure, and coincides with the initial attachment reaction between the trophectoderm and luminal epithelium. Vasoactive PGs are implicated in these processes. Here we demonstrate that COX genes are differentially regulated in the peri-implantation mouse uterus. During the preimplantation period (days 1–4), the COX-1 gene was expressed in the uterine epithelium mainly on day 4 until the initiation of attachment reaction in the evening after which the expression was downregulated. This COX-1 expression coincides with the generalized uterine edema required for luminal closure. In contrast, the COX-2 gene was expressed in the luminal epithelium and subepithelial stromal cells at the anti-mesometrial pole exclusively surrounding the blastocyst at the time of attachment reaction on day 4 and persisted through the morning of day 5. This uterine gene was not expressed at the sites of blastocyst apposition during progesterone (P4) treated delayed implantation, but was readily induced in the uterus surrounding the activated blastocysts after termination of the delay by estradiol-17β (E2). The results suggest that PG synthesis catalyzed by COX-2 is important for localized increased uterine vascular permeability and attachment reaction. The COX-1 gene that was downregulated from the time of attachment reaction on day 4 was again expressed in the mesometrial and anti-mesometrial secondary decidual beds on days 7 and 8. These results suggest that PGs generated by COX-1 are involved in decidualization and/or continued localized endometrial vascular permeability observed during this period. In contrast, the COX-2 gene, expressed at the anti-mesometrial pole on days 4 and 5, switched its expression to the mesometrial pole from day 6 onward. These results suggest that PGs produced at this site by COX-2 are involved in angiogenesis for the establishment of placenta. In the ovariectomized mice, the COX-1 gene was induced in the epithelium by a combined treatment with P4 and E2. However, P4 and/or E2 treatments failed to influence the uterine COX-2 gene. Overall, the results suggest that the uterine COX-1 gene is influenced by ovarian steroids, while the COX-2 gene is regulated by the implanting blastocyst during early pregnancy.
FY Diao, M Xu, Y Hu, J Li, Z Xu, M Lin, L Wang, Y Zhou, Z Zhou, J Liu, and J Sha
Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders; it is characterized by polycystic ovaries, hyperandrogenism and chronic anovulation. To obtain a global view of those genes that might be involved in the development of this complex clinical disorder, we used recently developed cDNA microarray technology to compare differential gene expressions between normal human ovary and ovaries from PCOS patients. A total of 9216 clones randomly selected from a commercial human ovary cDNA library were screened. Among them, 290 clones showed differential expressions, including 119 known genes and 100 known or unknown expressed sequence tags (ESTs). Among 119 known genes, 88 were upregulated and 31 downregulated in the PCOS ovary, as compared with normal human ovary. These differentially expressed genes are involved in various biologic functions, such as cell division/apoptosis, regulation of gene expression and metabolism, reflecting the complexity of clinical manifestations of PCOS. The molecular characteristics established from our study will further our understanding of the pathogenesis of PCOS and help us to identify new targets for further studies and for the development of new therapeutic interventions.
Sarah X Zhang, Joshua J Wang, Guoquan Gao, Kyoungmin Parke, and Jian-xing Ma
It has been shown that the balance between vascular endothelial growth factor (VEGF), a major angiogenic stimulator, and pigment epithelium-derived factor (PEDF), a potent angiogenic inhibitor, is critical for the regulation of vascular permeability and angiogenesis. However, the regulation of the balance is largely unclear. The present study demonstrated that there is a reciprocal interaction between VEGF and PEDF in the retina. PEDF significantly decreased VEGF expression in both retinal capillary endothelial cells (RCEC) and Müller cells. This PEDF effect was confirmed in the retina of rats with oxygen-induced retinopathy. Silencing of the PEDF gene by siRNA in Müller cells resulted in a significant upregulation of VEGF expression at both the RNA and protein levels, suggesting that PEDF is an endogenous negative regulator of VEGF. The further study of the mechanism showed that PEDF inhibited hypoxia-induced increases in VEGF promoter activity, HIF-1 nuclear translocation and mitogen activated protein kinase phosphorylation. These results suggest that PEDF inhibits VEGF expression at the transcriptional level. In addition, PEDF effectively inhibited VEGF binding to RCEC. Moreover, in vitro receptor-binding assay demonstrated that PEDF competed with VEGF for binding to VEGF receptor 2, which may represent a new mechanism for PEDF activity. On the other hand, VEGF significantly downregulated PEDF expression in RCEC, but not in retinal Müller cells, suggesting a VEGF receptor-mediated process. These results suggest that the reciprocal regulation between VEGF and PEDF may play a role in angiogenic control. The decrease in PEDF levels in the retina is at least partially responsible for the increase in VEGF expression and subsequent vascular leakage and neovascularization in diabetes.
S K Das, H Lim, J Wang, B C Paria, M BazDresch, and S K Dey
In the mouse, the initiation of the attachment reaction between the blastocyst trophectoderm and luminal epithelium of the receptive uterus occurs in the evening (2200-2300 h) of day 4 of pregnancy (day 1=vaginal plug) and is followed by proliferation and differentiation of stromal cells into decidual cells at the sites of blastocyst attachment. This investigation demonstrates that an inappropriate expression of the human transforming growth factor α (hTGF-α) transgene in the uterus under the direction of a mouse metallothionein-I promoter downregulates uterine expression of TGF-β receptor subtypes and delays the initiation of implantation (attachment reaction) resulting in delayed parturition. This delay in the attachment reaction is accompanied by deferred uterine expression of amphiregulin. The results suggest that a coordinated 'cross-talk' between the signaling pathways executed by epidermal growth factor-like growth factors and TGF-βs is important for the normal implantation process.
M.-W. Wang, A. Whyte, R. B. Heap, and M. J. Taussig
Passive immunization with a mouse monoclonal antibody against progesterone, designated DB3, blocks pregnancy in several species. We have previously reported that DB3 localizes in the mouse uterine epithelium shortly before normal implantation. This phenomenon is pregnancy dependent and specific for the progesterone antibody. In this study we demonstrate that DB3 is present in the lumen of the uterus 36 h after an i.p. injection; this correlates with the time of maximum antibody reaction on the uterine epithelium. Incubation of DB3 with free progesterone, progesterone-hemisuccinate or progesterone—bovine serum albumin before administration prevented its localization on the epithelium, indicating that the localization requires free progesterone-binding sites and thus probably depends upon progesterone binding. In addition, studies in vitro show that DB3 can effectively bind to progesterone carried by high-affinity progesterone-binding protein purified from coypu plasma. We suggest that specific targeting of DB3 may be through progesterone associated with a progesterone-binding molecule on the membrane of the uterine epithelia. This may be an important part of the mechanism of antibody action against implantation.