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K M Waters

Publications and source records attributed to K M Waters.

11 recordsLinked to original sources
PubMed2001

Differential gene expression in response to methoxychlor and estradiol through ERalpha, ERbeta, and AR in reproductive tissues of female mice.

The reproductive and developmental effects of 17beta-estradiol (E2) and methoxychlor (MXC) observed in treated rodents appear to be linked to some unique but also overlapping patterns of gene expression. The MXC metabolite 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) was previously shown to have selective agonist activity through estrogen receptor alpha (ERalpha) and antagonist activity through ERbeta and androgen receptor (AR). To discover gene families regulated by HPTE and E2, and to characterize similarities and differences in patterns of gene expression induced by these selective ER ligands, we analyzed tissues from mice treated for 3 days with a combined treatment of E2 and HPTE (E2 + HPTE), or the antiandrogen flutamide (FLU). RNA from uteri and ovaries was analyzed with cDNA microarrays and real-time RT-PCR. Results indicate that HPTE and E2 acted similarly to regulate most gene families in the uterus, which expresses predominantly ERalpha. However, in both the uterus and the ovary, there were a few genes that displayed differential patterns of gene regulation by E2 or HPTE treatment, presumably through ERbeta, AR, or other unidentified pathways. In the uterus, progesterone receptor, ERalpha, AR, insulin-like growth factor 1, insulin-like growth factor binding protein 5, and clusterin mRNAs were significantly reduced with both E2 or HPTE treatments, whereas cathepsin B was induced. Conversely, in the ovary, induction of cathepsin B by E2 was reversed after cotreatment with HPTE, and ERbeta expression was induced similarly by HPTE and FLU but not by E2. In addition, E2 uniquely regulated glutathione peroxidase 3, glutathione S-transferase, and cytochrome P450 17alpha-hydroxylase, with no effect of HPTE or FLU treatments. This analysis demonstrated several gene families that appear to be regulated in a ligand-specific pattern, which may explain the unique but overlapping reproductive tissue pathologies following exposure to E2 and MXC.

Animals↗
PubMed2000

Overexpression of a nuclear protein, TIEG, mimics transforming growth factor-beta action in human osteoblast cells.

Although transforming growth factor-beta (TGF-beta) is a growth factor with many known regulatory activities in many different cell types, its intracellular signaling pathway is still not fully understood. A TGF-beta-inducible early gene (TIEG) was discovered and shown by this laboratory to be a 3-zinc finger transcription factor family member; its expression is rapidly induced in cells treated with TGF-beta. To ascertain whether TIEG plays a major role in the TGF-beta pathway, human osteosarcoma MG-63 cells were stably transfected either with an expression vector containing a TIEG cDNA or with the vector alone. Clones that contain only the vector express normal levels of TIEG mRNA and protein and display the same patterns of gene expression and levels of cell proliferation as the nontransfected, non-TGF-beta-treated parental cells. However, transfected cells that overexpress TIEG mRNA and protein (TIEG-6 and TIEG-7) display changes that mimic those of MG-63 cells treated with TGF-beta, i.e. increased alkaline phosphatase activity, decreased levels of osteocalcin mRNA and protein, and decreased cell proliferation. The degree of these changes correlated with the level of TIEG expressed in the cell lines. TGF-beta treatment of the overexpressed cells showed no added effects. These findings and other published reports support a primary role of TIEG as a transcription factor in the TGF-beta signaling pathway.

Alkaline Phosphatase↗
PubMed2000

Direct action of naturally occurring estrogen metabolites on human osteoblastic cells.

This article describes experiments that were performed to examine the direct action of estrogen metabolites on cultured human osteoblast cells. The human fetal osteoblastic cell line, hFOB/ER9, which expresses high levels of the estrogen receptor (ER) alpha, was used to examine the direct effects of 16alpha-hydroxyestrone (16alpha-OHE1) and 2-hydroxyestrone (2-OHE1) on osteoblast differentiation. The 16alpha-OHE1 caused a decrease in osteocalcin (OC) secretion to a maximum of 40% of control values (vehicle-treated cells) at 10(-7) M. Alkaline phosphatase (AP) activity was significantly induced at 10(-7) M 16alpha-OHE1 with greater than 500% of control at 10(-6) M 16alpha-OHE1. Finally, AP steady-state messenger RNA (mRNA) levels were increased within 24 h of 16alpha-OHE1 treatment. In contrast to 16alpha-OHE1, 2-OHE1 had no effects on the secretion of OC, AP activity, or AP gene expression. The 2-OHE1 also did not display any antiestrogen activity because treatment in combination with 17beta-estradiol (E2) and 16alpha-OHE1 had no significant effect on the reduction in OC secretion or induction of AP activity. Similar to E2, 16alpha-OHE1 stimulated the expression of an early response gene, a TGF-beta inducible early gene, designated TIEG, as early as 60 minutes after treatment, whereas treatment with 2-OHE1 displayed no effect. Support that the 16alpha-OHE1 regulation of these osteoblasts (OB) markers was mediated through the ER is shown by the fact that the estrogen antagonist ICI 182,780 abrogated these effects. These data suggest that is a potent estrogen agonist on human osteoblastic hOB/ER9 cells. In contrast, 2-OHE1 displayed no estrogenic or antiestrogenic activity in this human osteoblast cell model.

Alkaline Phosphatase↗
PubMed1998

Estrogen regulation of a transforming growth factor-beta inducible early gene that inhibits deoxyribonucleic acid synthesis in human osteoblasts.

This laboratory reported the identification and characterization of a unique three zinc finger, transcription factor-like, transforming growth factor-beta inducible early gene (TIEG) (see Ref. 35). TIEG expression has been shown to be tissue- and cell type specific, enhanced by specific growth factors, and to decrease with advancing stages of breast cancer. Recent studies involving TIEG overexpression in pancreatic carcinoma cells indicate that TIEG expression inhibits DNA synthesis, similar to a tumor suppressor-like gene, and plays a role in apoptosis (see Ref. 37). This paper describes the rapid, but transient, induction of TIEG steady-state messenger RNA (mRNA) levels by 17beta-estradiol (E2) in estrogen receptor (ER)-positive, human fetal osteoblastic (hFOB/ER) cells. This rapid induction is shown to be ER- and steroid dose-dependent but protein synthesis independent. An antagonism between E2 and PTH, which occurs in skeletal metabolism, is shown to concur rapidly with TIEG mRNA expression. Scanning confocal microscopy (using polarized, laser-based immunofluorescence) shows that TIEG protein is localized in the nucleus of hFOB/ER cells, with the levels rapidly increasing after E2 treatment. The rapid E2-induced increase in TIEG expression is followed by an E2-induced inhibition of DNA synthesis in the hFOB/ER cells. Antiestrogens block not only the induction of TIEG mRNA levels but also the inhibition of cell proliferation. Lastly, hFOB cells, stably transfected with a TIEG expression vector, display markedly reduced DNA synthesis/cell proliferation, compared with nontransfected cells. These results support the finding that TIEG is an early responding regulatory gene for E2 in human osteoblast cells that inhibits DNA synthesis. It is speculated that TIEG may play a role in the signaling pathway for E2 in inhibiting cell proliferation.

Cell Division↗
PubMed1997

Localization of a polyunsaturated fatty acid response region in stearoyl-CoA desaturase gene 1.

Polyunsaturated fatty acids (PUFA) repress stearoyl-CoA desaturase gene 1 (SCD1) expression in liver and adipose tissues. We used HepG2 cells to localize genetic regulatory elements for PUFA in the SCD1 5'-flanking region. A chimeric reporter gene construct containing the 4.3 kb SCD1 putative promoter was transiently transfected into HepG2 cells, which were then treated with various fatty acids. We observed greater than 60% repression of transcription with 18:3n - 3 and 75% repression with 20:4n - 6 and 20:5n - 3. No significant change was seen with 18:0. Using smaller SCD1 chimeric constructs, we localized the genetic regulatory region to a 237 bp sequence within the SCD1 proximal promoter. DNA mobility shift analysis with HepG2 and mouse liver nuclear extracts demonstrated specific binding of nuclear proteins to this region. Mobility shift analysis with nuclear extract from 3T3-L1 adipocytes showed a similar pattern of protein binding. Competitive DNA mobility shift analysis identified a 60 bp region containing sites that specifically bind and compete for nuclear proteins. This region conferred responsiveness to PUFA when placed in a heterologous promoter. A homologous region in the stearoyl-CoA desaturase gene 2 (SCD2) promoter also mediated PUFA-specific repression in transfection experiments. These data suggest that a common transcriptional mechanism may exist in liver and adipose tissues for inhibition of lipogenesis by PUFA.

Adipocytes↗
PubMed1997

Localization of a negative thyroid hormone-response region in hepatic stearoyl-CoA desaturase gene 1.

The effect of thyroid hormone on stearoyl-CoA desaturase gene 1 (SCD1) expression was investigated in mouse liver. Daily injections of 15 micrograms triiodothyronine (T3)/100 g body weight to hypothyroid mice resulted in repression of SCD1 mRNA levels by more than 50% in 48 hours and up to 65% in 6 days. Transient co-transfections were performed with an expression vector for T3 receptor alpha (T3R alpha) in HepG2 cells using chimeric reporter gene constructs of the SCD1 5'-flanking region. Transcriptional repression of the SCD1 putative promoter was observed upon treatment with 100 nM T3 when cotransfected with T3R alpha, but not without cotransfection of receptor. Transient gene expression studies localized a T3 response region to a 70-bp sequence in the SCD1 putative promoter. Eliminating the TATA box and an AP-2 binding site, DNA mobility shift analysis demonstrated specific binding of in vivo nuclear protein from mouse liver nuclear extract to a 43-bp sequence. DNA mobility shift with purified T3R alpha confirmed the presence of a T3 receptor binding site in this thyroid hormone-responsive region. These data indicate that SCD1 contains a negative T3 response region in its proximal promoter.

Animals↗
PubMed1997

Regulation of hepatic stearoyl-CoA desaturase gene 1 by vitamin A.

The effect of vitamin A supplementation on stearoyl-CoA desaturase gene 1 expression in mouse liver was characterized. Normal BALB/c mice were fed 0.01% and 0.1% retinol palmitate as components of nonpurified diets. This treatment resulted in a 3-fold and a 7-fold induction of SCD1 mRNA levels, respectively, as determined by RNase protection analysis. Vitamin A-deficient animals were also fed diets containing 0.01% and 0.1% retinol palmitate, resulting in a similar pattern of SCD1 mRNA induction. Fatty acid synthase and beta-actin mRNA levels did not respond consistently or significantly to retinoic acid treatment. Dietary and hormonal studies were carried out to investigate the role of the retinoid X receptor in the regulation of SCD1 by type II steroid hormones. A receptor-saturating dose of thyroid hormone, triiodothyronine, repressed vitamin A-elevated SCD1 mRNA levels in vivo. Peroxisome proliferator-elevated SCD1 mRNA levels were unaffected by administration of thyroid hormone. This suggests that the retinoic acid receptor transcriptionally regulates SCD1 through a traditional mechanism of heterodimerization with the retinoid X receptor.

Actins↗
PubMed1996

Polyunsaturated fatty acids inhibit hepatic stearoyl-CoA desaturase-1 gene in diabetic mice.

Insulin and dietary fructose independently induce stearoyl-CoA desaturase 1 (SCD1) gene expression in diabetic mouse liver. In the present study, we again used diabetic mice and supplemented a high fructose diet with polyunsaturated fatty acids (PUFA) to determine the selective repression of SCD1 gene expression by dietary PUFA, as previously shown in normal mice. We saw dramatic repression of SCD1 mRNA expression, with trilinolenin at 3% and triarachidonin at 1% supplementation. We also observed significant repression of insulin-induced SCD1 mRNA upon supplementation of the noninducing starch diet with PUFA. In conclusion, our data demonstrate that PUFA negatively regulate hepatic gene expression through an insulin-independent mechanism.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1994

Insulin and dietary fructose induce stearoyl-CoA desaturase 1 gene expression of diabetic mice.

The transcription and mRNA levels of murine liver stearoyl-CoA desaturase 1 (SCD1) are induced 11- and 45-fold, respectively, by feeding fasted normal mice with a fat-free, high carbohydrate diet (Ntambi, J. M. (1992) J. Biol. Chem. 267, 10925-10930). In this study, we used streptozotocin-induced diabetic mice to study the regulatory role of carbohydrate and insulin on expression of the SCD1 gene in liver. Fructose administration to fasted diabetic mice induced a 2-fold increase in SCD1 mRNA within 6 h and a 23-fold increase within 24 h. Similarly, insulin administration to diabetic mice induced SCD1 mRNA from 4-fold within 4 h to 22-fold within 24 h. Insulin plus fructose, however, achieved full induction, with a 45-fold increase of SCD1 mRNA and a 10-fold increase in SCD1 transcription within 24 h. Additionally, the effect of insulin on SCD1 mRNA was inhibited 75% with dibutyryl-cAMP and theophylline administration and 70% by cycloheximide administration. Synthesis of liver albumin mRNA showed little change upon dietary manipulation or insulin treatment. Our data demonstrate that insulin and dietary fructose or a metabolite of fructose positively regulate the expression of the SCD1 gene in mouse liver.

Animals↗
PubMedDate not supplied

Estrogen regulation of human osteoblast function is determined by the stage of differentiation and the estrogen receptor isoform.

Although osteoblasts have been shown to respond to estrogens and express both isoforms of the estrogen receptor (ER alpha and ER beta), the role each isoform plays in osteoblast cell function and differentiation is unknown. The two ER isoforms are known to differentially regulate estrogen-inducible promoter-reporter gene constructs, but their individual effects on endogenous gene expression in osteoblasts have not been reported. We compared the effects of 17 beta-estradiol (E) and tamoxifen (TAM) on gene expression and matrix formation during the differentiation of human osteoblast cell lines stably expressing either ER alpha (hFOB/ER alpha 9) or ER beta (hFOB/ER beta 6). Expression of the appropriate ER isoform in these cells was confirmed by northern and western blotting and the responses to E in the hFOB/ER beta 6 line were abolished by an ER beta-specific inhibitor. The data demonstrate that (1) in both the hFOB/ER cell lines, certain responses to E or TAM (including alkaline phosphatase, IL-6 and IL-11 production) are more pronounced at the late mineralization stage of differentiation compared to earlier stages, (2) E exerted a greater regulation of bone nodule formation and matrix protein/cytokine production in the ER alpha cells than in ER beta cells, and (3) the regulated expression of select genes differed between the ER alpha and ER beta cells. TAM had no effect on nodule formation in either cell line and was a less potent regulator of gene/protein expression than E. Thus, both the ER isoform and the stage of differentiation appear to influence the response of osteoblast cells to E and TAM.

Alkaline Phosphatase↗