Basic transcription element binding (BTEB) protein is a newly identified member of the C 2 H 2 zi... more Basic transcription element binding (BTEB) protein is a newly identified member of the C 2 H 2 zinc finger family that also includes the transcription factors Spl, Sp2, Sp3, and Sp4. This family of proteins binds GC-rich motifs widely distributed in gene promoters, resulting in distinct activation or repression of transcriptional activities. Whereas Sp proteins are ubiquitously expressed, expression of BTEB appears more limited and has not been documented in the female reproductive tract of any mammalian species. This study was designed to identify and characterize the cellular distribution of BTEB in the porcine endometrium and placenta at known stages of pregnancy. Northern analysis of uterine endometrium detected BTEB mRNA that corresponds in size (5 kilobases) to that of the major BTEB transcript in rat brain. The steady-state levels of BTEB mRNA were higher (p < 0.05) in endometrium than placenta at corresponding days of pregnancy, although for each tissue, the levels did not change with pregnancy stage (p > 0.05). Luminal epithelial (LE), glandular epithelial (GE), and stromal (ST) cells isolated from pregnancy endometrium expressed the BTEB gene, but mRNA abundance varied with cell type (LE, GE > ST). Western blot analysis using an antiserum generated against the N-terminal region of a porcine BTEB fusion protein produced in Escherichia coli revealed the presence of BTEB protein only in endometrium, not in placenta. Immunohistochemical studies localized BTEB predominantly to the nuclei of endometrial GE and LE cells. Consistent with the presence of functional BTEB protein, binding to a double-stranded oligonucleotide containing multiple GC motifs was demonstrated in nuclear extracts prepared from endometrium and from endometrial LE and GE, but not ST, cells by electrophoretic mobility shift assay. These results demonstrate the preferential endometrial cell-type expression of BTEB and suggest its regulatory role in pregnancy-associated endometrial epithelial gene expression.
Malic enzyme 1 (ME1) is a cytosolic protein that catalyzes the conversion of malate to pyruvate w... more Malic enzyme 1 (ME1) is a cytosolic protein that catalyzes the conversion of malate to pyruvate while concomitantly generating NADPH from NADP. Early studies identified ME1 as a mediator of intermediary metabolism primarily through its participatory roles in lipid and cholesterol biosynthesis. ME1 was one of the first identified insulin-regulated genes in liver and adipose and is a transcriptional target of thyroxine. Multiple studies have since documented that ME1 is pro-oncogenic in numerous epithelial cancers. In tumor cells, the reduction of ME1 gene expression or the inhibition of its activity resulted in decreases in proliferation, epithelial-to-mesenchymal transition and in vitro migration, and conversely, in promotion of oxidative stress, apoptosis and/or cellular senescence. Here, we integrate recent findings to highlight ME1's role in oncogenesis, provide a rationale for its nexus with metabolic syndrome and diabetes, and raise the prospects of targeting the cytosolic NADPH network to improve therapeutic approaches against multiple cancers.
Two isozymes of porcine aromatase, the placental and the blastocyst forms, were expressed in CHO ... more Two isozymes of porcine aromatase, the placental and the blastocyst forms, were expressed in CHO cells using the mammalian cell transfection method. Using an `in-cell' assay (a 3 H-water release method), catalytic parameters of the porcine placental aromatase were found to be very similar to those of the human enzyme; however, the activity of the blastocyst isozyme was found to be one-thirtieth that of the placental isozyme. Product isolation assay (using testosterone as the substrate) revealed that the major steroid products were 17b-estradiol and 19-nortestosterone. The product ratio of estradiol/19-nortestosterone was found to be 94 : 6 for the porcine placental form, 6 : 94 for the porcine blastocyst form, and 92 : 8 for the human wild-type aromatase. Therefore, the porcine blastocyst aromatase isozyme catalyzes mainly androgen 19-desmethylation rather than aromatization. In addition, inhibition profile analyses on the placental and blastocyst isozymes were performed using three steroidal inhibitors [4-hydroxyandro-stenedione (4-OHA), 7a-(4 H -amino)phenylthio-1,4-androstandiene-3,17-dione (7a-APTADD), and bridge (2,19-methyleneoxy) androstene-3,17-dione (MDL 101,003)], and four nonsteroidal inhibitors [aminoglutethimide (AG), CGS 20267, ICI D1033, and vorozole (R83842)]. While the two isozymes of porcine aromatase share 93% amino-acid sequence identity, our results indicate that the two porcine aromatase isozymes have distinct responses to various aromatase inhibitors.
Expression of the transcription factor Krüppel-like factor 9 (KLF9) is frequently reduced in colo... more Expression of the transcription factor Krüppel-like factor 9 (KLF9) is frequently reduced in colorectal cancers, although a tumor suppressive role has not been established. To determine if KLF9 suppresses intestinal adenoma formation, we generated mice of distinct Klf9 genotypes in the background of the Apc Min/+ mouse and compared their adenoma burdens at 16 weeks of age. While small intestine adenoma burden remained unchanged among Klf9 genotypes, male and female
This study was designed to evaluate the ontogeny of serum IGF-I (SI) concentrations and its relat... more This study was designed to evaluate the ontogeny of serum IGF-I (SI) concentrations and its relationship to animal performance in a 140-d postweaning feeding trial. Ninety-eight progeny representing six sires (three high and three low feed conversion) and two sexes (43 bulls and 55 heifers) with ad libitum access to feed were allocated by sire and sex to monitor individual weights and pen feed consumption. Blood serum samples were obtained at the beginning of test (average age of 230 d) and every 28 d thereafter until each animal reached a fat thickness (estimated by sonoray) of 8.9 mm. Individual serum samples were acid-ethanol extracted and measured for IGF-I peptide by heterologous RIA. Serum IGF-I concentrations differed (P less than .10) between high (H) and low (L) feed conversion progeny groups at the end of the first 28-d period (125.12 vs 89.52 ng/ml) and tended to differ at the conclusion of the second 28-d period (P less than .15). Weight gains of H and L groups tended to differ in the second and third 28-d periods (P = .11 and .10, respectively). Serum IGF-I concentrations differed (P less than .05) between bulls and heifers for the first through fourth 28-d periods (P less than .01, P less than .05, P less than .10 and P less than .01, respectively). Phenotypic correlations indicated that pens with higher mean SI concentrations at the beginning of the test consumed less feed and had lower cumulative feed:gain ratios.(ABSTRACT TRUNCATED AT 250 WORDS)
Evidence is presented for the involvement of a number of specific uterine- and conceptus-derived ... more Evidence is presented for the involvement of a number of specific uterine- and conceptus-derived proteins in endometrial differentiation and conceptus or fetal development. These secretory proteins include mitogens (insulin-like growth factor-I and -II, epidermal growth factor, uterine luminal fluid mitogen), binding and transport proteins (uteroferrin, insulin-like growth factor and retinol binding proteins, respectively), protease inhibitors (antileukoproteinase, plasmin/trypsin inhibitor), and trophoblastic specific proteins. Using immunological reagents and specific complementary DNA (cDNA) probes, the tissue origins of several of these proteins have now been identified. In addition, the temporal regulation of messenger RNA (mRNA) production for a number of these proteins has been elucidated. The results suggest that although circulating and locally produced steroid hormones may be involved in regulating the synthetic abilities of these tissues during pregnancy, other, as yet undefined, factors may also mediate these activities. In this paper we present a review of the current knowledge pertaining to the identity, physiological regulation and potential functions of pig maternal and conceptus secretory proteins during pregnancy.
International Journal of Molecular Sciences, Apr 1, 2023
Malic Enzyme 1 (ME1) supports lipogenesis, cholesterol synthesis, and cellular redox potential by... more Malic Enzyme 1 (ME1) supports lipogenesis, cholesterol synthesis, and cellular redox potential by catalyzing the decarboxylation of L-malate to pyruvate, and the concomitant reduction of NADP to NADPH. We examined the contribution of ME1 to the development of obesity by provision of an obesogenic diet to C57BL/6 wild type (WT) and MOD-1 (lack ME1 protein) female mice. Adiposity, serum hormone levels, and adipose, mammary gland, liver, and small intestine gene expression patterns were compared between experimental groups after 10 weeks on a diet. Relative to WT female mice, MOD-1 female mice exhibited lower body weights and less adiposity; decreased concentrations of insulin, leptin, and estrogen; higher concentrations of adiponectin and progesterone; smaller-sized mammary gland adipocytes; and reduced hepatosteatosis. MOD-1 mice had diminished expression of Lep gene in abdominal fat; Lep, Pparg, Klf9, and Acaca genes in mammary glands; Pparg and Cdkn1a genes in liver; and Tlr9 and Ffar3 genes in the small intestine. By contrast, liver expression of Cdkn2a and Lepr genes was augmented in MOD-1, relative to WT mice. Results document an integrative role for ME1 in development of female obesity, suggest novel linkages with specific pathways/genes, and further support the therapeutic targeting of ME1 for obesity, diabetes, and fatty liver disease.
Pig blastocysts exhibit a transient period of estrogen production at periimplantation, with a sec... more Pig blastocysts exhibit a transient period of estrogen production at periimplantation, with a second, more sustained period of estrogen synthesis occurring in endometrium and placenta at later pregnancy. Previously we reported the isolation of cDNA clones encoding a novel isoform of cytochrome P450 aromatase (the terminal enzyme in the estrogen biosynthetic pathway) from porcine periimplantation blastocysts. The present study investigated pregnancy-associated expression, in blastocysts and maternal reproductive tract tissues, of this and an additional mRNA transcript encoding a distinct P450 aromatase isoform. Restriction endonuclease and nucleotide sequence analyses of 44 cDNA clones demonstrated that the major aromatase mRNA in periimplantation blastocysts and early-pregnancy endometrium and placenta (blastocyst-type) differed in sequence from the major aromatase mRNA expressed in endometrium and placenta at midpregnancy, (endometrial-type). The deduced blastocyst and endometrial aromatase isoform protein sequences had 93% similarity. A third type of aromatase mnRNA, deleted in exons 4-6 sequences, also was cloned from blastocysts. This cDNA was identical in nucleotide sequence to the blastocyst full-length aromatase cDNA and specified an open reading frame of 354 amino acids for a putative aromatase-related protein containing the heme-binding domain. Expression of this shorter mRNA in blastocysts was confirmed by reverse transcription polymerase chain reaction. The 5'-untranslated exon sequences in the transcripts encoding the blastocyst-type aromatase isoform were distinct from that of the endometrial type, consistent with differential expression of multiple chromosomal genes. In periimplantation equine embryos, however, embryonic and placental 5'untranslated exon-containing transcripts were coexpressed. Results identify an aromatase isoform expressed in the endometrium and placenta at midpregnancy, demonstrate a transition in synthesis of aromatase isoform-specific mRNAs during placental development, and suggest the preferential involvement of the blastocyst aromatase isoform in synthesis of estrogenic molecules that may function in embryo-maternal signaling at periimplantation.
The effects of supplementation of dietary clofibric acid (.5% wt/wt) on fatty acid binding protei... more The effects of supplementation of dietary clofibric acid (.5% wt/wt) on fatty acid binding protein (FABP) activity, apparent lipid digestibility, and serum cholesterol concentrations were evaluated in weanling pigs. Twenty-four barrows were allotted by weight and litter to a randomized complete block design with two treatments (basal vs clofibric acid) in six replicates. Nutrient digestibility measurements were made for a 2-wk period, after which the pigs were killed and tissues were collected. No differences in BW, liver, proximal small intestine, distal small intestine, and proximal and distal intestinal mucosa weights were observed. Apparent lipid digestibility was greater (P &lt; .05) for the overall 2-wk period in clofibric acid-supplemented pigs (81.5 vs 76.6%). This paralleled the increased FABP activity in the distal small intestine (P &lt; .001) of clofibric acid-supplemented pigs. Proximal intestine and liver FABP activities were unaffected by dietary treatment. Serum cholesterol concentrations were markedly lowered by clofibric acid supplementation. During wk 1, pigs fed the basal diet had twofold greater (P &lt; .01) serum cholesterol concentrations, whereas during wk 2, basal-fed pigs had fourfold greater (P &lt; .01) serum cholesterol concentrations (81.5 vs 18.3 mg/dL). These results suggest that elevated intestinal FABP activities may augment fatty acid absorption from the gastrointestinal tract.
Newborn pigs (n = 20) were gavage-fed sow’s colostrum, defatted colostrum, milk, defatted milk or... more Newborn pigs (n = 20) were gavage-fed sow’s colostrum, defatted colostrum, milk, defatted milk or a 5% lactose solution over 24 h in order to evaluate effects on growth and functional differentiation of small intestine. Colostrum-fed pigs had greater (p &lt; 0.01) mucosal mass in the proximal half of the small intestine than did the milk- or lactose-fed groups. Total fatty acid binding protein (FABP) activity and FABP activity per mg DNA in proximal intestines of colostrum-fed pigs exceeded that for the lactose group. FABP activities (per g mucosa or mg soluble protein) were greater (p &lt; 0.01) in the proximal segments of small intestines of pigs fed whole versus the corresponding defatted secretion. These results indicate that the feeding of colostrum specifically augments perinatal intestinal growth and differentiation as manifested by increased cellular hypertrophy and FABP activity. Milk lipid and unidentified factor(s) enriched in colostrum are inducers of intestinal FABP activity.
Porcine colostrum and milk were orally administered to newborn pigs to evaluate their gastrointes... more Porcine colostrum and milk were orally administered to newborn pigs to evaluate their gastrointestinal growth-promoting activity. Five piglets per treatment group were gavagefed 5% lactose Q, defatted colostrum (C) or defatted mature milk (d 16 of lactation) (M) at 3-h intervals over a 24-h period. Colostrum and milk were administered on equivalent dry matter basis and all piglets received 1 mCi of [3KJthymidine per kg B W at the onset of feeding. Small intestines of C-and M-fed pigs were 1.6-and 1.3-fold, respectively, the weight of small intestines of those fed L (P < .01). Total DNA content of intact small intestines were not different among groups; however, cpm [3H]thymidine/mg intestinal DNA of C and M piglets exceeded (P .05) that for L piglets. DNA content and cprn [3H]thymidine of intestinal mucosa did not differ among groups. Total protein in the intestines and intestinal mucosa of C pigs exceeded (P < .01) that for L and M pigs. Total RNA in the small intestine and intestinal mucosa were similar for C and M groups but less (P < .01) for L piglets. Stomach and pancreas weights among all pigs were similar, although C and M pigs exceeded L pigs in stomach (P c .01) and pancreas (P < .01) RNA content. In contrast, no differences in stomach DNA, protein and cpm [3KJthymidine or in pancreatic DNA, protein and cpm [3KJthymidine were detected. SDS-polyacrylamide gel electrophoresis was used to identify qualitative and quantitative differences in the protein compositions of porcine colostrum and mature milk, Feeding of either colostrum or mature milk was effective in promoting perinatal gut development (as reflected by RNA content) of pigs.
Estrogen, acting through its cognate receptor estrogen receptora (ESR1), is a critical regulator ... more Estrogen, acting through its cognate receptor estrogen receptora (ESR1), is a critical regulator of uterine endometrial epithelial proliferation. Although the dynamic communication between endometrial stromal (ST) and epithelial cells is considered to be an important component in this process, key molecular players in particular compartments remain poorly defined. Here, we used mice null for Kru ¨ppel-like factor 9 (KLF9) to evaluate the contribution of this nuclear protein in ST-epithelial interactions underlying proliferative effects of estrogen. We found that in ovariectomized mice administered estradiol-17b (E 2 ) for 24 h, Klf9 null mutation resulted in lack of E 2 -induced proliferative response in all endometrial compartments. We demonstrated a negative association between Klf9 expression and nuclear levels of ESR1 transcriptional corepressor prohibitin (PHB) 2 in uterine STand epithelial cells of E 2 -treated wild-type (WT) and Klf9 null mice. In early pregnancy uteri of WT mice, the temporal pattern of Klf9 transcript levels was inversely associated with that of Phb2. Deletion of Klf9 up-regulated uterine Phb2 expression and increased PHB2 nuclear localization in endometrial ST and epithelial cells, with no effects on the expression of the related Phb1. In the human endometrial ST cell line treated with E 2 for 24 h, Klf9 siRNA targeting augmented PHB2 transcript and increased nuclear PHB2 protein levels, albeit this effect was not to the extent seen in vivo with Klf9 null mutants. Our findings suggest a novel mechanism for control of estrogen-induced luminal epithelial proliferation involving ST KLF9 regulation of paracrine factor(s) to repress epithelial expression of corepressor PHB2.
Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL An increasing body of ev... more Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL An increasing body of evidence implicates obesity and the attendant hyperinsulinemia as major risk factors for human colorectal cancers. The lipogenic pathway supports growth and survival of colon cancer cells. In particular, inhibition of Fatty Acid Synthase (FASN) or Acetyl-CoA Carboxylase α (ACCα) induces apoptosis of colon cancer cells. The relative involvement in colon cancer initiation, progression and pathogenesis of cytosolic NADPH that is used in lipogenesis remains less clear. In the present study, we have determined the effects of insulin and the involvement of the major NADPH-generating cytosolic enzymes on proliferation of human colon cancer cell lines. Insulin (1-10 nM; to mimic normo- to hyper-insulinemic states) induced a significant increase (7-15%, p < 0.05) in proliferation of HT-29 and HCT-116 colon cancer cell lines in vitro (assayed by MTT assay). Addition of insulin augmented (by 5-10%) proliferation of both cell lines upon concurrent treatment with cancer chemotherapeutic agents (5-fluorouracil, oxaliplatin, irinotecan; each added at a dose < EC50). The insulin-mediated increase in HT-29 cell growth was associated with a 2-3 fold elevation in FASN gene expression and a 50% decrease in expression of the FASN-targeting micro RNAs, miR-103 and miR-107. However, insulin did not induce mRNA abundance for the cytoplasmic NADPH-generating enzymes [Malic Enzyme 1 (ME1), Glucose 6-Phosphate Dehydrogenase (G6PD), 6-Phosphogluconate Dehydrogenase (6PGD), or Isocitrate Dehydrogenase-1 (IDH1]. siRNA-mediated knockdown of FASN or of the individual major cytosolic NADPH-generating enzymes caused decreases in proliferation of HT-29 and HCT-116 cells (15-30%, p < 0.05) compared to the control scrambled siRNA. Data highlight pro-proliferative roles for insulin, FASN and cytosolic NADPH-generating enzymes in colorectal cancer cell growth. Implementation of pharmacologic and/or nutritional means to reduce serum insulin levels, enhance FASN miR expression and inhibit tumor cell NADPH-generation may serve as useful adjuncts for future cancer treatment and prevention. Supported by NIH grant RO1 CA136493 and the Arkansas Biosciences Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4072. doi:10.1158/1538-7445.AM2011-4072
Tile goal of this study was to characterize and quantify" intestinal filtty acid-binding proteins... more Tile goal of this study was to characterize and quantify" intestinal filtty acid-binding proteins of the pig. Small intestinal mucosa from 13-19 kg pigs was homogenized and centr~f'uged to obtain cytosol. Isolation o f'.~ltty acid-binding proteins .f)'om delipidated ~Ttosol was achieved using molecular sieve, olei(" acid aJfinity, and ion exchange chromatography. Fatty acid-binding protein isolation u'as monitored using a fatty-aeid binding assay in coniunetion with sodium dodeevl sMl2~te-polyaeo'htmide gel electrophoresis and immam~blotting. Antisera to rat liver-l~ltty acid-binding protein eros.s reacted with an isolated intestinal fatty acid-binding protein of M, -13,000, wherea,~ antisera to rat intestine-l~tty acid-binding protein was trot cross reactive with isolated pig intestinal proteins. These experinlents identify a pig, intestinal fatty acid-binding protein lhal exhibits strong immunoehemical similarity to rat liver-.fatty acid-binding protein. ()'tosol prepared from intestinal nlueosa of pigs at -4. 2, 4, 7, 15.22, 28, attd 35 d of age was assayed Jbr [}ttO, acid-binding protein activiO'. Preweaning.[~*tty acidbinding protein activio, in cytosol was maximal at 7 days of age when expressed as totaljejunalj~ttty acid binding pet kilogram hodyweight, intestinal or maeosal weight or milligram total protein. After weaning (21 d), fatty acid-binding protein aetivitie.s declined to 28 days, hut increased again by 35 days. Total soluble .fatty acid-binding protein aetivio' in pig intestine is regtdated during postnatal development and this may account in part fin" the altered intestinal absorption of lipids observed in 3,olttlg pigs at weaning.
Pig blastocysts exhibit a transient period of estrogen production at periimplantation, with a sec... more Pig blastocysts exhibit a transient period of estrogen production at periimplantation, with a second, more sustained period of estrogen synthesis occurring in endometrium and placenta at later pregnancy. Previously we reported the isolation of cDNA clones encoding a novel isoform of cytochrome P450 aromatase (the terminal enzyme in the estrogen biosynthetic pathway) from porcine periimplantation blastocysts. The present study investigated pregnancy-associated expression, in blastocysts and maternal reproductive tract tissues, of this and an additional mRNA transcript encoding a distinct P450 aromatase isoform. Restriction endonuclease and nucleotide sequence analyses of 44 cDNA clones demonstrated that the major aromatase mRNA in periimplantation blastocysts and early-pregnancy endometrium and placenta (blastocyst-type) differed in sequence from the major aromatase mRNA expressed in endometrium and placenta at midpregnancy, (endometrial-type). The deduced blastocyst and endometrial aromatase isoform protein sequences had 93% similarity. A third type of aromatase mnRNA, deleted in exons 4-6 sequences, also was cloned from blastocysts. This cDNA was identical in nucleotide sequence to the blastocyst full-length aromatase cDNA and specified an open reading frame of 354 amino acids for a putative aromatase-related protein containing the heme-binding domain. Expression of this shorter mRNA in blastocysts was confirmed by reverse transcription polymerase chain reaction. The 5'-untranslated exon sequences in the transcripts encoding the blastocyst-type aromatase isoform were distinct from that of the endometrial type, consistent with differential expression of multiple chromosomal genes. In periimplantation equine embryos, however, embryonic and placental 5'untranslated exon-containing transcripts were coexpressed. Results identify an aromatase isoform expressed in the endometrium and placenta at midpregnancy, demonstrate a transition in synthesis of aromatase isoform-specific mRNAs during placental development, and suggest the preferential involvement of the blastocyst aromatase isoform in synthesis of estrogenic molecules that may function in embryo-maternal signaling at periimplantation.
The coexpression of IGF (-I and -II) peptides, corresponding receptors, and IGF binding proteins ... more The coexpression of IGF (-I and -II) peptides, corresponding receptors, and IGF binding proteins (IGFBPs) in uterine endometrium suggests that a significant component of IGF action in this tissue is via autocrine or paracrine pathways, or both. The present study examined whether IGF-II and a major uterine-expressed IGF-II binding protein, IGFBP-2, modulate endometrial epithelial cell mitogenesis. Serum-deprived porcine endometrial glandular epithelial (GE) cells of early pregnancy were treated with various concentrations of IGFs, recombinant porcine (rp) IGFBP-2, or both, and examined for changes in cellular mitogenesis by incorporation of [ 3 H]thymidine into DNA. Recombinant human (rh) IGF-II stimulated DNA synthesis in a dose-dependent manner. Human [Leu 27 ]-IGF-II, an analog with selective affinity for the IGF-II (type II) receptor, increased thymidine uptake by twofold compared with untreated GE cells. When added in combination with an equimolar concentration of rhIGF-I, [Leu 27 ]-IGF-II or rhIGF-II stimulated thymidine incorporation to a greater extent than did rhIGF-I alone. Ligand blot analysis of GE cell conditioned medium revealed the presence of four IGFBPs with molecular masses of 48, 31, 23, and 15 kDa. Physiological concentrations of rpIGFBP-2 (nM range) increased both basal and IGF-induced DNA synthesis in GE cells. At equimolar concentrations, Des(1-6)IGF-II (an IGF-II analog with much reduced affinity for IGFBPs) and rpIGFBP-2 had additive effects on GE cell mitogenesis, suggesting that the IGFBP-2 modulation of uterine cell growth may involve both IGF-dependent and IGF-independent pathways. Our results demonstrate the complex interplay of IGF system components in uterine endometrial epithelial growth regulation in vitro, identify IGF-II and IGFBP-2 as locally coexpressed uterine epithelial cell mitogens, and suggest the presence of a functional signaling pathway by which IGF-II stimulates epithelial cell proliferation via the type II receptor.
Despite the well‐accepted notion of peri‐natal origins of adult diseases, the factors and regulat... more Despite the well‐accepted notion of peri‐natal origins of adult diseases, the factors and regulatory mechanisms underlying breast cancer development remain unclear. Diet is a highly modifiable determinant of breast cancer risk, and the effects of the in utero nutritional environment persist beyond fetal life. We investigated whether in utero/lactational exposure to blueberry (BB) via maternal diet alters the trajectory of Wnt1‐induced mammary tumorigenesis in offspring. Wnt1 transgenic mice were exposed to maternal diets of casein (CAS; n=33) or blueberry‐supplemented CAS (3% BB; n=28) from gestation day 4 until post‐natal day 21. Offspring were then weaned to CAS and mammary tumor development was followed until age 8 months. While tumor incidence and latency were similar for both groups, tumor weight (by 2‐fold, p=0.034) and growth rate (by 60%; p=0.008) were reduced in offspring of BB‐ versus CAS‐fed dams. Tumors from the BB group had higher expression of tumor suppressors PTEN an...
Basic transcription element binding (BTEB) protein is a newly identified member of the C 2 H 2 zi... more Basic transcription element binding (BTEB) protein is a newly identified member of the C 2 H 2 zinc finger family that also includes the transcription factors Spl, Sp2, Sp3, and Sp4. This family of proteins binds GC-rich motifs widely distributed in gene promoters, resulting in distinct activation or repression of transcriptional activities. Whereas Sp proteins are ubiquitously expressed, expression of BTEB appears more limited and has not been documented in the female reproductive tract of any mammalian species. This study was designed to identify and characterize the cellular distribution of BTEB in the porcine endometrium and placenta at known stages of pregnancy. Northern analysis of uterine endometrium detected BTEB mRNA that corresponds in size (5 kilobases) to that of the major BTEB transcript in rat brain. The steady-state levels of BTEB mRNA were higher (p < 0.05) in endometrium than placenta at corresponding days of pregnancy, although for each tissue, the levels did not change with pregnancy stage (p > 0.05). Luminal epithelial (LE), glandular epithelial (GE), and stromal (ST) cells isolated from pregnancy endometrium expressed the BTEB gene, but mRNA abundance varied with cell type (LE, GE > ST). Western blot analysis using an antiserum generated against the N-terminal region of a porcine BTEB fusion protein produced in Escherichia coli revealed the presence of BTEB protein only in endometrium, not in placenta. Immunohistochemical studies localized BTEB predominantly to the nuclei of endometrial GE and LE cells. Consistent with the presence of functional BTEB protein, binding to a double-stranded oligonucleotide containing multiple GC motifs was demonstrated in nuclear extracts prepared from endometrium and from endometrial LE and GE, but not ST, cells by electrophoretic mobility shift assay. These results demonstrate the preferential endometrial cell-type expression of BTEB and suggest its regulatory role in pregnancy-associated endometrial epithelial gene expression.
Malic enzyme 1 (ME1) is a cytosolic protein that catalyzes the conversion of malate to pyruvate w... more Malic enzyme 1 (ME1) is a cytosolic protein that catalyzes the conversion of malate to pyruvate while concomitantly generating NADPH from NADP. Early studies identified ME1 as a mediator of intermediary metabolism primarily through its participatory roles in lipid and cholesterol biosynthesis. ME1 was one of the first identified insulin-regulated genes in liver and adipose and is a transcriptional target of thyroxine. Multiple studies have since documented that ME1 is pro-oncogenic in numerous epithelial cancers. In tumor cells, the reduction of ME1 gene expression or the inhibition of its activity resulted in decreases in proliferation, epithelial-to-mesenchymal transition and in vitro migration, and conversely, in promotion of oxidative stress, apoptosis and/or cellular senescence. Here, we integrate recent findings to highlight ME1's role in oncogenesis, provide a rationale for its nexus with metabolic syndrome and diabetes, and raise the prospects of targeting the cytosolic NADPH network to improve therapeutic approaches against multiple cancers.
Two isozymes of porcine aromatase, the placental and the blastocyst forms, were expressed in CHO ... more Two isozymes of porcine aromatase, the placental and the blastocyst forms, were expressed in CHO cells using the mammalian cell transfection method. Using an `in-cell' assay (a 3 H-water release method), catalytic parameters of the porcine placental aromatase were found to be very similar to those of the human enzyme; however, the activity of the blastocyst isozyme was found to be one-thirtieth that of the placental isozyme. Product isolation assay (using testosterone as the substrate) revealed that the major steroid products were 17b-estradiol and 19-nortestosterone. The product ratio of estradiol/19-nortestosterone was found to be 94 : 6 for the porcine placental form, 6 : 94 for the porcine blastocyst form, and 92 : 8 for the human wild-type aromatase. Therefore, the porcine blastocyst aromatase isozyme catalyzes mainly androgen 19-desmethylation rather than aromatization. In addition, inhibition profile analyses on the placental and blastocyst isozymes were performed using three steroidal inhibitors [4-hydroxyandro-stenedione (4-OHA), 7a-(4 H -amino)phenylthio-1,4-androstandiene-3,17-dione (7a-APTADD), and bridge (2,19-methyleneoxy) androstene-3,17-dione (MDL 101,003)], and four nonsteroidal inhibitors [aminoglutethimide (AG), CGS 20267, ICI D1033, and vorozole (R83842)]. While the two isozymes of porcine aromatase share 93% amino-acid sequence identity, our results indicate that the two porcine aromatase isozymes have distinct responses to various aromatase inhibitors.
Expression of the transcription factor Krüppel-like factor 9 (KLF9) is frequently reduced in colo... more Expression of the transcription factor Krüppel-like factor 9 (KLF9) is frequently reduced in colorectal cancers, although a tumor suppressive role has not been established. To determine if KLF9 suppresses intestinal adenoma formation, we generated mice of distinct Klf9 genotypes in the background of the Apc Min/+ mouse and compared their adenoma burdens at 16 weeks of age. While small intestine adenoma burden remained unchanged among Klf9 genotypes, male and female
This study was designed to evaluate the ontogeny of serum IGF-I (SI) concentrations and its relat... more This study was designed to evaluate the ontogeny of serum IGF-I (SI) concentrations and its relationship to animal performance in a 140-d postweaning feeding trial. Ninety-eight progeny representing six sires (three high and three low feed conversion) and two sexes (43 bulls and 55 heifers) with ad libitum access to feed were allocated by sire and sex to monitor individual weights and pen feed consumption. Blood serum samples were obtained at the beginning of test (average age of 230 d) and every 28 d thereafter until each animal reached a fat thickness (estimated by sonoray) of 8.9 mm. Individual serum samples were acid-ethanol extracted and measured for IGF-I peptide by heterologous RIA. Serum IGF-I concentrations differed (P less than .10) between high (H) and low (L) feed conversion progeny groups at the end of the first 28-d period (125.12 vs 89.52 ng/ml) and tended to differ at the conclusion of the second 28-d period (P less than .15). Weight gains of H and L groups tended to differ in the second and third 28-d periods (P = .11 and .10, respectively). Serum IGF-I concentrations differed (P less than .05) between bulls and heifers for the first through fourth 28-d periods (P less than .01, P less than .05, P less than .10 and P less than .01, respectively). Phenotypic correlations indicated that pens with higher mean SI concentrations at the beginning of the test consumed less feed and had lower cumulative feed:gain ratios.(ABSTRACT TRUNCATED AT 250 WORDS)
Evidence is presented for the involvement of a number of specific uterine- and conceptus-derived ... more Evidence is presented for the involvement of a number of specific uterine- and conceptus-derived proteins in endometrial differentiation and conceptus or fetal development. These secretory proteins include mitogens (insulin-like growth factor-I and -II, epidermal growth factor, uterine luminal fluid mitogen), binding and transport proteins (uteroferrin, insulin-like growth factor and retinol binding proteins, respectively), protease inhibitors (antileukoproteinase, plasmin/trypsin inhibitor), and trophoblastic specific proteins. Using immunological reagents and specific complementary DNA (cDNA) probes, the tissue origins of several of these proteins have now been identified. In addition, the temporal regulation of messenger RNA (mRNA) production for a number of these proteins has been elucidated. The results suggest that although circulating and locally produced steroid hormones may be involved in regulating the synthetic abilities of these tissues during pregnancy, other, as yet undefined, factors may also mediate these activities. In this paper we present a review of the current knowledge pertaining to the identity, physiological regulation and potential functions of pig maternal and conceptus secretory proteins during pregnancy.
International Journal of Molecular Sciences, Apr 1, 2023
Malic Enzyme 1 (ME1) supports lipogenesis, cholesterol synthesis, and cellular redox potential by... more Malic Enzyme 1 (ME1) supports lipogenesis, cholesterol synthesis, and cellular redox potential by catalyzing the decarboxylation of L-malate to pyruvate, and the concomitant reduction of NADP to NADPH. We examined the contribution of ME1 to the development of obesity by provision of an obesogenic diet to C57BL/6 wild type (WT) and MOD-1 (lack ME1 protein) female mice. Adiposity, serum hormone levels, and adipose, mammary gland, liver, and small intestine gene expression patterns were compared between experimental groups after 10 weeks on a diet. Relative to WT female mice, MOD-1 female mice exhibited lower body weights and less adiposity; decreased concentrations of insulin, leptin, and estrogen; higher concentrations of adiponectin and progesterone; smaller-sized mammary gland adipocytes; and reduced hepatosteatosis. MOD-1 mice had diminished expression of Lep gene in abdominal fat; Lep, Pparg, Klf9, and Acaca genes in mammary glands; Pparg and Cdkn1a genes in liver; and Tlr9 and Ffar3 genes in the small intestine. By contrast, liver expression of Cdkn2a and Lepr genes was augmented in MOD-1, relative to WT mice. Results document an integrative role for ME1 in development of female obesity, suggest novel linkages with specific pathways/genes, and further support the therapeutic targeting of ME1 for obesity, diabetes, and fatty liver disease.
Pig blastocysts exhibit a transient period of estrogen production at periimplantation, with a sec... more Pig blastocysts exhibit a transient period of estrogen production at periimplantation, with a second, more sustained period of estrogen synthesis occurring in endometrium and placenta at later pregnancy. Previously we reported the isolation of cDNA clones encoding a novel isoform of cytochrome P450 aromatase (the terminal enzyme in the estrogen biosynthetic pathway) from porcine periimplantation blastocysts. The present study investigated pregnancy-associated expression, in blastocysts and maternal reproductive tract tissues, of this and an additional mRNA transcript encoding a distinct P450 aromatase isoform. Restriction endonuclease and nucleotide sequence analyses of 44 cDNA clones demonstrated that the major aromatase mRNA in periimplantation blastocysts and early-pregnancy endometrium and placenta (blastocyst-type) differed in sequence from the major aromatase mRNA expressed in endometrium and placenta at midpregnancy, (endometrial-type). The deduced blastocyst and endometrial aromatase isoform protein sequences had 93% similarity. A third type of aromatase mnRNA, deleted in exons 4-6 sequences, also was cloned from blastocysts. This cDNA was identical in nucleotide sequence to the blastocyst full-length aromatase cDNA and specified an open reading frame of 354 amino acids for a putative aromatase-related protein containing the heme-binding domain. Expression of this shorter mRNA in blastocysts was confirmed by reverse transcription polymerase chain reaction. The 5'-untranslated exon sequences in the transcripts encoding the blastocyst-type aromatase isoform were distinct from that of the endometrial type, consistent with differential expression of multiple chromosomal genes. In periimplantation equine embryos, however, embryonic and placental 5'untranslated exon-containing transcripts were coexpressed. Results identify an aromatase isoform expressed in the endometrium and placenta at midpregnancy, demonstrate a transition in synthesis of aromatase isoform-specific mRNAs during placental development, and suggest the preferential involvement of the blastocyst aromatase isoform in synthesis of estrogenic molecules that may function in embryo-maternal signaling at periimplantation.
The effects of supplementation of dietary clofibric acid (.5% wt/wt) on fatty acid binding protei... more The effects of supplementation of dietary clofibric acid (.5% wt/wt) on fatty acid binding protein (FABP) activity, apparent lipid digestibility, and serum cholesterol concentrations were evaluated in weanling pigs. Twenty-four barrows were allotted by weight and litter to a randomized complete block design with two treatments (basal vs clofibric acid) in six replicates. Nutrient digestibility measurements were made for a 2-wk period, after which the pigs were killed and tissues were collected. No differences in BW, liver, proximal small intestine, distal small intestine, and proximal and distal intestinal mucosa weights were observed. Apparent lipid digestibility was greater (P &lt; .05) for the overall 2-wk period in clofibric acid-supplemented pigs (81.5 vs 76.6%). This paralleled the increased FABP activity in the distal small intestine (P &lt; .001) of clofibric acid-supplemented pigs. Proximal intestine and liver FABP activities were unaffected by dietary treatment. Serum cholesterol concentrations were markedly lowered by clofibric acid supplementation. During wk 1, pigs fed the basal diet had twofold greater (P &lt; .01) serum cholesterol concentrations, whereas during wk 2, basal-fed pigs had fourfold greater (P &lt; .01) serum cholesterol concentrations (81.5 vs 18.3 mg/dL). These results suggest that elevated intestinal FABP activities may augment fatty acid absorption from the gastrointestinal tract.
Newborn pigs (n = 20) were gavage-fed sow’s colostrum, defatted colostrum, milk, defatted milk or... more Newborn pigs (n = 20) were gavage-fed sow’s colostrum, defatted colostrum, milk, defatted milk or a 5% lactose solution over 24 h in order to evaluate effects on growth and functional differentiation of small intestine. Colostrum-fed pigs had greater (p &lt; 0.01) mucosal mass in the proximal half of the small intestine than did the milk- or lactose-fed groups. Total fatty acid binding protein (FABP) activity and FABP activity per mg DNA in proximal intestines of colostrum-fed pigs exceeded that for the lactose group. FABP activities (per g mucosa or mg soluble protein) were greater (p &lt; 0.01) in the proximal segments of small intestines of pigs fed whole versus the corresponding defatted secretion. These results indicate that the feeding of colostrum specifically augments perinatal intestinal growth and differentiation as manifested by increased cellular hypertrophy and FABP activity. Milk lipid and unidentified factor(s) enriched in colostrum are inducers of intestinal FABP activity.
Porcine colostrum and milk were orally administered to newborn pigs to evaluate their gastrointes... more Porcine colostrum and milk were orally administered to newborn pigs to evaluate their gastrointestinal growth-promoting activity. Five piglets per treatment group were gavagefed 5% lactose Q, defatted colostrum (C) or defatted mature milk (d 16 of lactation) (M) at 3-h intervals over a 24-h period. Colostrum and milk were administered on equivalent dry matter basis and all piglets received 1 mCi of [3KJthymidine per kg B W at the onset of feeding. Small intestines of C-and M-fed pigs were 1.6-and 1.3-fold, respectively, the weight of small intestines of those fed L (P < .01). Total DNA content of intact small intestines were not different among groups; however, cpm [3H]thymidine/mg intestinal DNA of C and M piglets exceeded (P .05) that for L piglets. DNA content and cprn [3H]thymidine of intestinal mucosa did not differ among groups. Total protein in the intestines and intestinal mucosa of C pigs exceeded (P < .01) that for L and M pigs. Total RNA in the small intestine and intestinal mucosa were similar for C and M groups but less (P < .01) for L piglets. Stomach and pancreas weights among all pigs were similar, although C and M pigs exceeded L pigs in stomach (P c .01) and pancreas (P < .01) RNA content. In contrast, no differences in stomach DNA, protein and cpm [3KJthymidine or in pancreatic DNA, protein and cpm [3KJthymidine were detected. SDS-polyacrylamide gel electrophoresis was used to identify qualitative and quantitative differences in the protein compositions of porcine colostrum and mature milk, Feeding of either colostrum or mature milk was effective in promoting perinatal gut development (as reflected by RNA content) of pigs.
Estrogen, acting through its cognate receptor estrogen receptora (ESR1), is a critical regulator ... more Estrogen, acting through its cognate receptor estrogen receptora (ESR1), is a critical regulator of uterine endometrial epithelial proliferation. Although the dynamic communication between endometrial stromal (ST) and epithelial cells is considered to be an important component in this process, key molecular players in particular compartments remain poorly defined. Here, we used mice null for Kru ¨ppel-like factor 9 (KLF9) to evaluate the contribution of this nuclear protein in ST-epithelial interactions underlying proliferative effects of estrogen. We found that in ovariectomized mice administered estradiol-17b (E 2 ) for 24 h, Klf9 null mutation resulted in lack of E 2 -induced proliferative response in all endometrial compartments. We demonstrated a negative association between Klf9 expression and nuclear levels of ESR1 transcriptional corepressor prohibitin (PHB) 2 in uterine STand epithelial cells of E 2 -treated wild-type (WT) and Klf9 null mice. In early pregnancy uteri of WT mice, the temporal pattern of Klf9 transcript levels was inversely associated with that of Phb2. Deletion of Klf9 up-regulated uterine Phb2 expression and increased PHB2 nuclear localization in endometrial ST and epithelial cells, with no effects on the expression of the related Phb1. In the human endometrial ST cell line treated with E 2 for 24 h, Klf9 siRNA targeting augmented PHB2 transcript and increased nuclear PHB2 protein levels, albeit this effect was not to the extent seen in vivo with Klf9 null mutants. Our findings suggest a novel mechanism for control of estrogen-induced luminal epithelial proliferation involving ST KLF9 regulation of paracrine factor(s) to repress epithelial expression of corepressor PHB2.
Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL An increasing body of ev... more Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL An increasing body of evidence implicates obesity and the attendant hyperinsulinemia as major risk factors for human colorectal cancers. The lipogenic pathway supports growth and survival of colon cancer cells. In particular, inhibition of Fatty Acid Synthase (FASN) or Acetyl-CoA Carboxylase α (ACCα) induces apoptosis of colon cancer cells. The relative involvement in colon cancer initiation, progression and pathogenesis of cytosolic NADPH that is used in lipogenesis remains less clear. In the present study, we have determined the effects of insulin and the involvement of the major NADPH-generating cytosolic enzymes on proliferation of human colon cancer cell lines. Insulin (1-10 nM; to mimic normo- to hyper-insulinemic states) induced a significant increase (7-15%, p < 0.05) in proliferation of HT-29 and HCT-116 colon cancer cell lines in vitro (assayed by MTT assay). Addition of insulin augmented (by 5-10%) proliferation of both cell lines upon concurrent treatment with cancer chemotherapeutic agents (5-fluorouracil, oxaliplatin, irinotecan; each added at a dose < EC50). The insulin-mediated increase in HT-29 cell growth was associated with a 2-3 fold elevation in FASN gene expression and a 50% decrease in expression of the FASN-targeting micro RNAs, miR-103 and miR-107. However, insulin did not induce mRNA abundance for the cytoplasmic NADPH-generating enzymes [Malic Enzyme 1 (ME1), Glucose 6-Phosphate Dehydrogenase (G6PD), 6-Phosphogluconate Dehydrogenase (6PGD), or Isocitrate Dehydrogenase-1 (IDH1]. siRNA-mediated knockdown of FASN or of the individual major cytosolic NADPH-generating enzymes caused decreases in proliferation of HT-29 and HCT-116 cells (15-30%, p < 0.05) compared to the control scrambled siRNA. Data highlight pro-proliferative roles for insulin, FASN and cytosolic NADPH-generating enzymes in colorectal cancer cell growth. Implementation of pharmacologic and/or nutritional means to reduce serum insulin levels, enhance FASN miR expression and inhibit tumor cell NADPH-generation may serve as useful adjuncts for future cancer treatment and prevention. Supported by NIH grant RO1 CA136493 and the Arkansas Biosciences Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4072. doi:10.1158/1538-7445.AM2011-4072
Tile goal of this study was to characterize and quantify" intestinal filtty acid-binding proteins... more Tile goal of this study was to characterize and quantify" intestinal filtty acid-binding proteins of the pig. Small intestinal mucosa from 13-19 kg pigs was homogenized and centr~f'uged to obtain cytosol. Isolation o f'.~ltty acid-binding proteins .f)'om delipidated ~Ttosol was achieved using molecular sieve, olei(" acid aJfinity, and ion exchange chromatography. Fatty acid-binding protein isolation u'as monitored using a fatty-aeid binding assay in coniunetion with sodium dodeevl sMl2~te-polyaeo'htmide gel electrophoresis and immam~blotting. Antisera to rat liver-l~ltty acid-binding protein eros.s reacted with an isolated intestinal fatty acid-binding protein of M, -13,000, wherea,~ antisera to rat intestine-l~tty acid-binding protein was trot cross reactive with isolated pig intestinal proteins. These experinlents identify a pig, intestinal fatty acid-binding protein lhal exhibits strong immunoehemical similarity to rat liver-.fatty acid-binding protein. ()'tosol prepared from intestinal nlueosa of pigs at -4. 2, 4, 7, 15.22, 28, attd 35 d of age was assayed Jbr [}ttO, acid-binding protein activiO'. Preweaning.[~*tty acidbinding protein activio, in cytosol was maximal at 7 days of age when expressed as totaljejunalj~ttty acid binding pet kilogram hodyweight, intestinal or maeosal weight or milligram total protein. After weaning (21 d), fatty acid-binding protein aetivitie.s declined to 28 days, hut increased again by 35 days. Total soluble .fatty acid-binding protein aetivio' in pig intestine is regtdated during postnatal development and this may account in part fin" the altered intestinal absorption of lipids observed in 3,olttlg pigs at weaning.
Pig blastocysts exhibit a transient period of estrogen production at periimplantation, with a sec... more Pig blastocysts exhibit a transient period of estrogen production at periimplantation, with a second, more sustained period of estrogen synthesis occurring in endometrium and placenta at later pregnancy. Previously we reported the isolation of cDNA clones encoding a novel isoform of cytochrome P450 aromatase (the terminal enzyme in the estrogen biosynthetic pathway) from porcine periimplantation blastocysts. The present study investigated pregnancy-associated expression, in blastocysts and maternal reproductive tract tissues, of this and an additional mRNA transcript encoding a distinct P450 aromatase isoform. Restriction endonuclease and nucleotide sequence analyses of 44 cDNA clones demonstrated that the major aromatase mRNA in periimplantation blastocysts and early-pregnancy endometrium and placenta (blastocyst-type) differed in sequence from the major aromatase mRNA expressed in endometrium and placenta at midpregnancy, (endometrial-type). The deduced blastocyst and endometrial aromatase isoform protein sequences had 93% similarity. A third type of aromatase mnRNA, deleted in exons 4-6 sequences, also was cloned from blastocysts. This cDNA was identical in nucleotide sequence to the blastocyst full-length aromatase cDNA and specified an open reading frame of 354 amino acids for a putative aromatase-related protein containing the heme-binding domain. Expression of this shorter mRNA in blastocysts was confirmed by reverse transcription polymerase chain reaction. The 5'-untranslated exon sequences in the transcripts encoding the blastocyst-type aromatase isoform were distinct from that of the endometrial type, consistent with differential expression of multiple chromosomal genes. In periimplantation equine embryos, however, embryonic and placental 5'untranslated exon-containing transcripts were coexpressed. Results identify an aromatase isoform expressed in the endometrium and placenta at midpregnancy, demonstrate a transition in synthesis of aromatase isoform-specific mRNAs during placental development, and suggest the preferential involvement of the blastocyst aromatase isoform in synthesis of estrogenic molecules that may function in embryo-maternal signaling at periimplantation.
The coexpression of IGF (-I and -II) peptides, corresponding receptors, and IGF binding proteins ... more The coexpression of IGF (-I and -II) peptides, corresponding receptors, and IGF binding proteins (IGFBPs) in uterine endometrium suggests that a significant component of IGF action in this tissue is via autocrine or paracrine pathways, or both. The present study examined whether IGF-II and a major uterine-expressed IGF-II binding protein, IGFBP-2, modulate endometrial epithelial cell mitogenesis. Serum-deprived porcine endometrial glandular epithelial (GE) cells of early pregnancy were treated with various concentrations of IGFs, recombinant porcine (rp) IGFBP-2, or both, and examined for changes in cellular mitogenesis by incorporation of [ 3 H]thymidine into DNA. Recombinant human (rh) IGF-II stimulated DNA synthesis in a dose-dependent manner. Human [Leu 27 ]-IGF-II, an analog with selective affinity for the IGF-II (type II) receptor, increased thymidine uptake by twofold compared with untreated GE cells. When added in combination with an equimolar concentration of rhIGF-I, [Leu 27 ]-IGF-II or rhIGF-II stimulated thymidine incorporation to a greater extent than did rhIGF-I alone. Ligand blot analysis of GE cell conditioned medium revealed the presence of four IGFBPs with molecular masses of 48, 31, 23, and 15 kDa. Physiological concentrations of rpIGFBP-2 (nM range) increased both basal and IGF-induced DNA synthesis in GE cells. At equimolar concentrations, Des(1-6)IGF-II (an IGF-II analog with much reduced affinity for IGFBPs) and rpIGFBP-2 had additive effects on GE cell mitogenesis, suggesting that the IGFBP-2 modulation of uterine cell growth may involve both IGF-dependent and IGF-independent pathways. Our results demonstrate the complex interplay of IGF system components in uterine endometrial epithelial growth regulation in vitro, identify IGF-II and IGFBP-2 as locally coexpressed uterine epithelial cell mitogens, and suggest the presence of a functional signaling pathway by which IGF-II stimulates epithelial cell proliferation via the type II receptor.
Despite the well‐accepted notion of peri‐natal origins of adult diseases, the factors and regulat... more Despite the well‐accepted notion of peri‐natal origins of adult diseases, the factors and regulatory mechanisms underlying breast cancer development remain unclear. Diet is a highly modifiable determinant of breast cancer risk, and the effects of the in utero nutritional environment persist beyond fetal life. We investigated whether in utero/lactational exposure to blueberry (BB) via maternal diet alters the trajectory of Wnt1‐induced mammary tumorigenesis in offspring. Wnt1 transgenic mice were exposed to maternal diets of casein (CAS; n=33) or blueberry‐supplemented CAS (3% BB; n=28) from gestation day 4 until post‐natal day 21. Offspring were then weaned to CAS and mammary tumor development was followed until age 8 months. While tumor incidence and latency were similar for both groups, tumor weight (by 2‐fold, p=0.034) and growth rate (by 60%; p=0.008) were reduced in offspring of BB‐ versus CAS‐fed dams. Tumors from the BB group had higher expression of tumor suppressors PTEN an...
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