Biomedical subjects

R C Simmen

Publications and source records attributed to R C Simmen.

At least 19 recordsLinked to original sources

PubMed2001

Increased expression of the Zn-finger transcription factor BTEB1 in human endometrial cells is correlated with distinct cell phenotype, gene expression patterns, and proliferative responsiveness to serum and TGF-beta1.

Basic transcription element binding (BTEB, also designated BTEB1) protein is a member of the Sp-family of GC-box binding transcription factors that exhibit distinct patterns of expression in many cell types and tissues. A role for BTEB1 in the regulation of cell growth and gene transcription has been invoked, but little is known about the molecular mechanisms underlying these activities. The present study examined the functional consequences of high and low BTEB1 expression in the human endometrial carcinoma cell line Hec-1-A, by deriving stable clonal lines that expressed sense (S) and anti-sense (As) rat BTEB1 constructs. Clonal S lines, with BTEB1 mRNA and protein levels higher than in corresponding parent (N) and As lines, displayed enhanced DNA synthesis upon 3[H]-thymidine incorporation, in serum-containing but not in serum-free medium, and increased cell cycle kinetics, concomitant with the induction in expression of the genes for the cell cycle-associated components cyclin D1, PCNA, cyclin-dependent kinase (Cdk) inhibitor p21, and Cdk2. Compared to N and As lines, S lines also had diminished ability to grow in multi-layers and exhibited increased mRNA levels for plasminogen activator inhibitor-1 (PAI-1), secretory leukocyte protease inhibitor (SLPI), and tissue inhibitor of metalloproteinases (TIMP)-2. In serum-free medium, S, but not N nor As lines, had enhanced DNA synthesis with transforming growth factor (TGF)-beta1, albeit all lines demonstrated similar responses to insulin-like growth factor-I and to epidermal growth factor, respectively. The higher DNA synthesis in S relative to N and As, lines upon exogenous TGF-beta1 addition, was observed in concert with increased expression of cyclins D1 and E and p21, genes. Moreover, S and As lines had increased mRNA levels for TIMP-1, TIMP-2, PAI-1, and beta-catenin, and diminished SLPI, and to a lesser extent, Cdk4 mRNA levels, with TGF-beta1 treatment. These results suggest that BTEB1 may mediate cell growth, in part, by modulating gene expression levels of distinct cell cycle and growth-associated proteins. The correlation between serum- and TGF-beta1 induction of DNA synthesis with increased BTEB1 expression further suggests that BTEB1 may constitute an important downstream regulatory component of various signaling pathways utilized by serum-associated and other growth factors in endometrial epithelial cells.

Animals↗

PubMed2001

Polyamine- and insulin-like growth factor-I-mediated proliferation of porcine uterine endometrial cells: a potential role for spermidine/spermine N(1)-acetyltransferase during peri-implantation.

Insulin-like growth factor-I (IGF-I) and the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) are progesterone-regulated genes with maximal expression at peri-implantation in the porcine uterine endometrium. However, while IGF-I stimulates cell proliferation, SSAT, by acetylating the naturally occurring polyamines (PA) spermine (SPM) and spermidine (SPD), typically functions as a cell growth inhibitor. The present study examined the functional relationships of IGF-I, SSAT, and PA in the control of endometrial cell proliferation. Northern blot analysis indicated that SSAT mRNA levels change with distinct pregnancy stages, in contrast to those for the PA biosynthetic enzyme ornithine decarboxylase (ODC). Primary cultures of luminal and glandular epithelial (LE, GE) and stromal (ST) cells isolated from Day 12 pregnant pig endometrium had IGF-I mRNA levels for ST > LE > GE cells. The mRNA levels for SSAT and ODC were transiently diminished by IGF-I treatment, but only in GE cells. By contrast, SPM and SPD increased SSAT mRNA levels in GE and ST cells, but increased ODC mRNA levels only in GE cells. IGF-I, putrescine (PUT), and SPM individually increased cellular DNA synthesis as measured by tritiated thymidine incorporation in GE and ST cells, while SPD had an effect only in ST cells. IGF-I enhanced the proliferative effect of each PA in GE cells, but only of SPD in ST cells. The mitogen-activated protein kinase inhibitor, PD98059, inhibited the induction by SPM of GE cell DNA synthesis but not that of IGF-I. Wortmannin, a phosphatidylinositol-3-kinase inhibitor had no effect on either IGF-I or SPM induction of GE cell DNA synthesis. The relative concentrations of SPM, SPD, and PUT in uterine luminal fluids differed, with the levels for each PA higher at pregnancy Day 12 than at 11.5. These results suggest that IGF-I and PA act through distinct signaling pathways to mediate cell-type-specific growth of early pregnancy pig uterine endometrium. Further, SSAT, through its control of intracellular PA levels, likely plays a modulatory role in the establishment of an optimal uterine environment for successful embryo attachment.

Acetylation↗

PubMed2001

Molecular cloning of the porcine acid-labile subunit (ALS) of the insulin-like growth factor-binding protein complex and detection of ALS gene expression in hepatic and non-hepatic tissues.

The acid-labile subunit (ALS) is an approximately 85 kDa N-glycoprotein that is known primarily as a component of the systemic insulin-like growth factor-binding protein (IGFBP) complex. We have amplified, using a PCR, three overlapping porcine ALS genomic DNA fragments that together encode the distal region of the signal peptide through to the COOH-terminus. The compiled sequence of 1775 nucleotides of the three overlapping DNAs and the deduced amino acid sequence of the mature porcine ALS (pALS) protein exhibited 84/81%, 79/77%, 79/78% and 84/79% identities with respect to those of the human, the rat, the mouse and the baboon respectively. Four conserved cysteine residues in the NH(2)-terminal domain and 20 leucine-rich repeats in the central domain also were identified at identical positions in the porcine ALS. By using Northern blot analysis, with a genomic DNA fragment as the probe, it was determined that a 2.2 kb ALS mRNA was induced in the liver during the late fetal stage, and hepatic ALS mRNA abundance was increased post-natally. Moreover, hepatic ALS mRNA abundance was increased by daily injection of porcine somatotropin (100 microg/kg body weight) in cross-bred market pigs each weighing approximately 100 kg. The ALS mRNA was not detected by Northern analysis in any non-hepatic tissue examined. However, results of a more sensitive solution hybridization/RNAse protection assay indicated that low levels of ALS mRNA were also present in adult muscle, spleen, ovary and uterus, but not in lung, kidney, oviduct and placenta. Taken together, the present results suggest that although liver is the primary organ that expresses the ALS gene under somatotropin stimulation, some non-hepatic tissues also express the gene at low levels in the pig.

Amino Acid Sequence↗

PubMed2001

Association of a genetic marker with blood serum insulin-like growth factor-I concentration and growth traits in Angus cattle.

The objective of this research was to evaluate a biallelic genetic marker identified in the first promoter region of the bovine IGF-I gene. The point mutation was identified as a T-to-C transition by sequencing the polymorphic fragments. A PCR-RFLP procedure was developed for determining the marker genotypes. Marker genotypes were determined for 760 Angus calves from divergent lines that were created by selection for high or low serum IGF-I concentration (allele A: 63.9%, B: 36.1%). Data were analyzed using the multiple-trait derivative-free restricted maximum likelihood computer programs with animal models. The full animal model included fixed effects of marker genotype, birth year, season of birth, sex, age of dam, and selection line; random effects of animal, maternal genetic, and maternal permanent environmental effects; and a covariate for age of calf. Traits analyzed included blood serum IGF-I concentrations on d 28, 42, and 56 of the postweaning test, mean IGF-I concentration, birth weight, weaning weight, on-test weight, off-test weight, off-test hip height, postweaning gain, and weight gain during the 20-d period immediately after weaning. Results from the analysis across selection lines showed a significant association of the BB genotype with higher weight gain during the first 20 d after weaning and a slight dominance effect of the marker on postweaning gain. Analysis within the low IGF-I line also showed a significant association of the BB genotype with higher weight gain during the first 20 d after weaning and with on-test weight, although analysis within the high IGF-I line did not show any significant association. The associated effects of the marker need to be verified in other cattle populations.

Animals↗

PubMed2000

Expression and regulatory function of the transcription factor Sp1 in the uterine endometrium at early pregnancy: implications for epithelial phenotype.

The uterus during early pregnancy synthesizes a complex array of signaling molecules with specific spatial and temporal modes of expression and which are critical for embryo implantation and subsequent development. The mechanism(s) underlying the differential pattern of synthesis of these pregnancy-associated proteins is not understood very well. The present study evaluated the expression and trans-activation potential of the transcription factor Sp1 in the early pregnancy porcine endometrium to determine its temporal and functional association with the endometrial epithelial-specific genes encoding the transplacental iron-transport protein uteroferrin (UF) and an Sp-family member, basic transcription element-binding (BTEB) protein. Two identical Sp1 clones (717 bp) were isolated from a porcine endometrial cDNA library by polymerase chain reaction (PCR). The nucleotide sequence of these clones encodes a partial protein sequence of 238 amino acids encompassing the Zn-finger region and had significant identities with the corresponding regions in the rat and human proteins. By using a specific antibody raised against human Sp1, porcine endometrial Sp1 was found to exhibit a molecular weight of 110 kDa, was localized predominantly in the nuclei of glandular and luminal epithelial cells, and appeared to exist as a phosphorylated protein. Northern blot analysis demonstrated three distinct size transcripts of approximately 3.5, 5, and 8 kb for endometrial Sp1. The expression of Sp1 mRNA and protein, determined by RT-PCR and by its ability to bind Sp1 consensus motif in gel mobility shift assays, respectively, overlapped with, but did not parallel that of UF mRNA during early pregnancy. The effect of increased Sp1 expression on UF gene promoter activity was examined using a human Sp1 expression vector that was transiently transfected into primary cultures of pig endometrial glandular epithelial cells. Sp1 increased (P < 0.05) the promoter activities of various UF promoter-Luciferase reporter constructs by 2 to 4-fold, over those transfected with empty expression vector. Co-transfection of a BTEB expression vector with the Sp1 expression vector modified the effect of Sp1 on UF promoter activity in the shortest construct. These results suggest that Sp1 mediates the regulation of endometrial epithelial gene expression during pregnancy, and that this function is likely altered in vivo by co-expression of other family members, including BTEB.

Acid Phosphatase↗

PubMed2000

Multiple isoforms of porcine aromatase are encoded by three distinct genes.

Cytochrome P450 aromatase, a product of the CYP 19 gene and the terminal enzyme in the estrogen biosynthetic pathway, is synthesized by the ovary, endometrium, placenta, and peri-implantation embryos in the pig and other mammals, albeit to varying levels, implying its functional role(s) in pregnancy events. The aromatase produced by the pig tissues exists as three distinct isoforms (type I - ovary, type II - placenta, and type III - embryo), with presumed differences in substrate specificities, expression levels, activity, and mode of regulation. In order to delineate the molecular mechanisms whereby estrogen synthesis is regulated in these diverse tissues, the present study examined if these aromatase isoforms represent products of multiple genes or of a single gene via complex splicing mechanisms. Porcine genomic DNA from a single animal was used as a template in the polymerase chain reaction (PCR) to amplify isoform-specific sequences corresponding to exons 4 and 7, respectively. Nucleotide sequence analysis of the generated fragments revealed the presence of only clones corresponding to the three known aromatase types. Screening a porcine Bacterial Artificial Chromosome (BAC) library for aromatase gene by PCR yielded a single clone approximately 80 kb in length. Southern blot analysis, using probes specific for exons 1A-1B, 2-3, 4-9, and 10 sequences indicated that the BAC genomic clone contains the entirety of the coding exons as well as the proximal promoter region. Sequence analysis of the fragment generated with exon 4 primers determined that this BAC clone contains only the type II gene. The presence and relative orientation of the untranslated 5'- exons 1A and 1B, previously demonstrated for the type III isoform were evaluated in the BAC clone and genomic DNA by PCR. The 265 bp fragment generated from both PCR reactions was confirmed by sequence analysis to contain exons 1A and 1B that are located contiguous to each other and separated by only three bp. A diagnostic procedure for typing aromatase isoforms was developed, based on the presence of specific restriction sites within isoform-specific exons. The use of this protocol confirmed the existence of only three aromatase isoforms in the porcine genome and indicated changes in aromatase types expressed by the uterine endometrium as a function of pregnancy stage. The presence of distinct genes encoding each of the aromatase isoform predicts important differences in the mechanisms underlying the molecular evolution and regulation of porcine aromatase, unique from those of other mammals, and suggests a critical role for P450 aromatase steroidal products in uterine functions related to pregnancy events.

Animals↗

PubMed2000

Pregnancy-dependent expression of leukaemia inhibitory factor (LIF), LIF receptor-beta and interleukin-6 (IL-6) messenger ribonucleic acids in the porcine female reproductive tract.

Leukaemia inhibitory factor (LIF) and interleukin-6 (IL-6) are candidate embryo-maternal signalling molecules which are present within the uterine luminal micro-environment. We examined the relative expression of the mRNAs encoding LIF and IL-6, as well as the LIF-binding subunit (LIFR-beta) of the LIF receptor and, as a potential downstream cytokine-responsive gene, beta(2)-microglobulin (beta(2)m), in porcine peri-implantation conceptuses, and in placenta and endometrium during early and mid-pregnancy. Peri-implantation spherical and filamentous conceptuses expressed LIFR-beta and beta(2)m mRNAs with no LIF mRNA present. Rapid development in days 11/12 spherical conceptuses to the filamentous stage was accompanied by transiently increased IL-6 gene expression. The corresponding endometrium, in contrast, expressed LIF in addition to these other mRNAs. LIFR-beta, IL-6 and beta(2)m, but not LIF mRNAs, were expressed in the Jag-1 cell line, an in vitro model for porcine day 14 trophoblast. The greatest steady-state amounts of LIF, LIFR-beta and IL-6 mRNAs in both the endometrium and placenta were evident at the post-implantation stages (days 30 and 60>day 18 of pregnancy). Treatment of porcine endometrial explants with human recombinant (hr)LIF or hrIL-6 resulted in no change in, or diminished, the presence of endometrial beta(2)m mRNA, respectively. Addition of LIF to peri-implantation conceptus explant cultures, in contrast, induced beta(2)m mRNA synthesis. These results highlight the potential importance of both the endometrium and placenta as sources, as well as targets, of these cytokines throughout pregnancy. Cytokine modulation of beta(2)m, a known in vitro mitogen, may constitute one mechanism for local control of trophoblast and endometrial proliferation.

Animals↗

PubMed2000

Genetic parameter estimates for serum insulin-like growth factor-I concentration and carcass traits in Angus beef cattle.

Divergent selection for serum insulin-like growth factor-I (IGF-I) concentration began at the Eastern Ohio Resource Development Center (EORDC) in 1989 using 100 spring-calving (50 high line and 50 low line) and 100 fall-calving (50 high line and 50 low line) purebred Angus cows. Following weaning, bull and heifer calves were fed in drylot for a 140-d postweaning period. At the conclusion of the postweaning test, bulls not selected for breeding were slaughtered and carcass data were collected at a commercial abbatoir. At the time of this analysis, IGF-I measurements were available for 1,283 bull and heifer calves, and carcass data were available for 452 bulls. A set of multiple-trait, derivative-free, restricted maximum likelihood (MTDFREML) computer programs were used for data analysis. Estimates of direct heritability for IGF-I concentration at d 28, 42, and 56 of the postweaning period, and for mean IGF-I concentration were .32, .59, .31, and .42, respectively. Direct heritabilities for carcass traits ranged from .27 to 1.0, .26 to 1.0, and .23 to 1.0 when the age-, fat-, and weight-constant end points, respectively, were used, with marbling score having the smallest heritability and longissimus muscle area having the highest heritability in each case. Maternal heritability and the proportion of phenotypic variance due to permanent environmental effect of dam generally were < or = .21 for IGF-I concentrations and for carcass traits other than longissimus muscle area. Additive genetic correlations of IGF-I concentrations with backfat thickness, longissimus muscle area, hot carcass weight, marbling score, quality grade, and yield grade averaged -.26, .19, -.04, -.53, -.45, and -.27, respectively, when carcass data were adjusted to an age-constant end point. Bulls with lower IGF-I concentrations had higher marbling scores and quality grades, but also had higher backfat thickness and yield grades regardless of the slaughter end point. Serum IGF-I concentration may be a useful selection criterion when efforts are directed toward improvement of marbling scores and quality grades of beef cattle.

Animals↗

PubMed1999

Uterine-associated serine protease inhibitors stimulate deoxyribonucleic acid synthesis in porcine endometrial glandular epithelial cells of pregnancy.

Protease inhibitors are major secretory components of the mammalian uterus that are thought to mediate pregnancy-associated events primarily by regulating the activity of proteolytic enzymes. In the present study, we examined the mitogenic potentials of two serine protease inhibitors, namely secretory leukocyte protease inhibitor (SLPI) and uterine plasmin/trypsin inhibitor (UPTI) in primary cultures of glandular epithelial (GE) cells isolated from early pregnant (Day 12) pig endometrium, using the [(3)H]thymidine incorporation assay. Purified porcine SLPI (pSPLI), porcine UPTI (pUPTI), or recombinant human SLPI (rhSLPI), all of which exhibited anti-trypsin activity, increased (p < 0.05) labeled thymidine incorporation into DNA of serum-deprived GE cells when tested at a range of 10-1000-ng/ml concentrations. Polyclonal antibodies directed against either hSLPI or pSLPI abrogated the effect of SLPI. Co-addition of pSLPI and pUPTI increased DNA synthesis in these cells to a level higher (p < 0.05) than that observed with either protease inhibitor. The glycosaminoglycan heparin, which has been previously shown to increase the anti-protease activity of SLPI, exhibited a tendency (p = 0.08) to enhance SLPI and UPTI induction of cellular DNA synthesis. Reverse transcription-polymerase chain reaction indicated that the messenger RNAs for both protease inhibitors were present in the endometrium throughout pregnancy and, within this tissue, in GE cells to a greater extent (p < 0.05) than in stromal fibroblastic cells. Results demonstrate that, in addition to their well-documented anti-protease activities, SLPI and UPTI may constitute autocrine growth promotants for the uterine epithelium. These data suggest a novel mechanism whereby locally produced protease inhibitors may modulate periimplantation events and embryo-maternal communication.

Animals↗

PubMed1999

Trans-activation functions of the Sp-related nuclear factor, basic transcription element-binding protein, and progesterone receptor in endometrial epithelial cells.

The present study examined the trans-activation potential of basic transcription element-binding protein (BTEB), a recently identified member of the Sp family of GC box-binding transcription factors, on the expression of the gene encoding the pregnancy-associated, epithelial-specific, and progesterone (P)-induced porcine uterine endometrial secretory protein, uteroferrin (UF). Endometrial expression of BTEB, P receptor (PR), and UF genes was analyzed by RT-PCR as a function of pregnancy stage and cell type and was correlated with the levels of endometrial BTEB that were quantified by Western blot and/or electrophoretic mobility shift assay. PR, BTEB, and UF messenger RNAs (mRNAs) were present in early (day 12) and mid(day 60) pregnancy pig endometrium, although expression levels varied for each mRNA (UF, day 12 << day 60; PR and BTEB, day 12 = day 60). Within the endometrium, glandular epithelial (GE) cells manifested higher amounts of UF mRNA than stromal fibroblastic cells, whereas both cell types had comparable amounts of BTEB and PR mRNAs. Expression of BTEB, however, was limited to endometrial GE cells. A BTEB expression vector (pcDNA-3BTEB) was used to examine the effect of increased BTEB protein on UF gene expression and promoter activity in primary cultures of pig endometrial GE cells. Cells transiently transfected with pcDNA-3BTEB had 2-fold higher UF mRNA levels than those transfected with the empty expression vector (pcDNA-3). Further, cells cotransfected with a UF promoter-luciferase (-1935UF-Luc) reporter gene and the BTEB expression vector had 2-fold higher Luc activity than those cotransfected with reporter gene and pcDNA-3. This effect of BTEB was not observed in transfected endometrial stromal fibroblastic cells, but was apparent in the human endometrial epithelial carcinoma cell lines ECC-1 and Hec-1-A, which exhibit low levels of BTEB protein and low or undetectable PR mRNA levels, respectively. The respective contributions of BTEB and PR to the modulation of UF promoter activity were examined by cotransfection of Hec-1-A and ECC-1 cells with expression plasmids for BTEB and PR and one of two UF promoter constructs (-831UF-Luc or -1935UF-Luc) in the absence or presence of P. The increase in UF promoter activity with BTEB was mimicked by PR in a P-dependent manner in both cell lines. The combined effect of PR/P and BTEB appeared additive in Hec-1-A cells and was synergistic in ECC-1 cells. These results highlight the cell context dependence of the trans-activation potential of BTEB and suggest its unique role, in concert with PR, in directing the temporal expression of endometrial epithelial genes of pregnancy.

Acid Phosphatase↗

PubMed1999

Complex mediation of uterine endometrial epithelial cell growth by insulin-like growth factor-II (IGF-II) and IGF-binding protein-2.

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.

Animals↗

PubMed1999

Reproductive performance of bulls divergently selected on the basis of blood serum insulin-like growth factor I concentration.

The objectives of this study were to examine differences in scrotal circumference, sperm motility, and percentage of normal sperm cells between two lines of Angus beef cattle divergently selected for blood serum IGF-I concentration. Data were obtained from an ongoing experiment involving 100 spring-calving (50 high and 50 low line) and 100 fall-calving (50 high and 50 low line) purebred Angus cows. Scrotal circumference, percentage of motile sperm cells, and percentage of normal sperm cells did not differ between high and low IGF-I line yearling bulls (P = .79, .50, and .56, respectively). The IGF-I concentrations measured at d 28, 42, and 56 of the postweaning test are abbreviated as IGF28, IGF42, and IGF56, respectively. Coefficients for the quadratic regression of scrotal circumference on IGF28 and IGF42 tended to be negative (P = .07 and .08, respectively), as did the coefficient for the quadratic regression of the percentage of motile sperm cells on IGF42 (P = .08). The coefficient for the linear regression of percentage of normal sperm cells on IGF28 was positive (P = .02). The coefficient for the quadratic regression of percentage of normal sperm cells on IGF56 was negative (P = .04). Coefficients for the quadratic regression of scrotal circumference and percentage of normal sperm cells on mean IGF-I concentrations were negative and important (P = .04 and .08, respectively). Thus, scrotal circumference and percentage of normal sperm cells are related to blood serum IGF-I concentration in yearling Angus bulls.

Animals↗

PubMed1998

The porcine insulin-like growth factor system: at the interface of nutrition, growth and reproduction.

The IGF system is implicated in the regulation of cellular response to protein- and energy-restriction. Although it is clear that the IGF and their binding proteins are profoundly influenced by dietary factors, a number of important questions remain about this relationship. In particular, although studies to date have focused on nutritional modulation of hepatic IGF gene expression, the molecular mechanisms underlying metabolic regulation of liver IGF and IGF binding protein genes remain relatively unknown. Moreover, the potential effects of altered nutrition on the expression and/or actions of IGF system components in tissues other than the liver have been examined only in cursory fashion. Many of these studies have used rats, an admittedly important model, but one which differs from the human in a potentially significant way: rats lack circulating IGF-II and IGFBP-2 during post-weaning and adult life. Here, we summarize current research on the porcine IGF system and highlight the particular usefulness this system may offer for unraveling the complex relationships of nutrition and systemic/local IGF expression and actions that are relevant to human nutritional physiology.

Amino Acid Sequence↗

PubMed1998

Control of secretory leukocyte protease inhibitor gene expression in the porcine periimplantation endometrium: a case of maternal-embryo communication.

The contributions of the conceptus (embryo and associated membranes) and of the maternal endocrine milieu to control of endometrial secretory leukocyte protease inhibitor (SLPI, also designated antileukoproteinase) expression during the periimplantation stages of embryo development were examined in the present study. Uterine endometrium from distinct sites was collected from pigs at Days 16-25 of pregnancy and analyzed for steady-state SLPI mRNA levels. Endometrium situated directly beneath conceptuses (mesometrial) had greater (p < 0.05) SLPI mRNA levels than that obtained from antimesometrial and interimplantation sites and myometrium. This site-specific difference was most pronounced during late (Days 19-21) and post (Days 23-25)-implantation stages and was also observed, albeit to a lesser degree, for the mRNA encoding uteroferrin (Uf). Conditioned medium (CM; 50% v:v) from Day 21, but not Day 12, conceptuses increased (p < 0.05) SLPI mRNA levels, while neither CM affected mRNA levels for Uf and several other genes expressed in endometrial explants. One inducing factor in Day 21 CM was characterized as a low-molecular-mass (< 12 kDa), relatively heat-stable protein. Transforming growth factor (TGF alpha) increased (p < 0.05), epidermal growth factor (EGF) tended to increase (p = 0.10), and insulin-like growth factor (IGF)-I and IGF-II had no effect (p > 0.10) on, SLPI mRNA levels in Day 12 endometrial explants. Medium conditioned by the pig trophoblast cell line, Jag-1, but not by other mammalian cell lines, had SLPI inducer activity. The maternal endocrine contribution to SLPI gene expression was examined using freshly isolated and short-term-cultured Day 12 and Day 21 pregnant pig endometrium. Steady-state SLPI mRNA levels were increased (p < 0.05) in Day 12, but not Day 21, tissues upon short-term culture in serum-free medium. This increase was time dependent, was similarly demonstrated for Uf mRNA, was not observed in corresponding lung and liver, and was inhibited by inclusion of serum from pigs of diverse endocrine status. In summary, two potential modulators of endometrial SLPI gene expression were identified: 1) a conceptus-derived low-molecular-mass protein, possibly TGF alpha, that mediates in part the up-regulation of SLPI gene expression in endometrium closely associated with implantation sites, and 2) an inhibitory component(s) present in maternal serum. Results suggest opposing actions of maternal and embryonic factors at the maternal-embryo interface and highlight involvement of the periimplantation embryo in directing the spatiotemporal expression of endometrial genes implicated in its development.

Animals↗

PubMed1998

Paracrine inducers of uterine endometrial spermidine/spermine N1-acetyltransferase gene expression during early pregnancy in the pig.

The endogenous factors that underlie the transient induction of the gene encoding spermidine/spermine N1-acetyltransferase (SSAT), the rate-limiting enzyme in cellular polyamine catabolism, in pig uterine endometrium during periimplantation are not known. The present study examined a number of peptide growth factors and regulatory molecules that are present within the uterine environment at early pregnancy, coincident with maximal SSAT gene expression, for their ability to manifest endogenous SSAT gene-inducing activity. Basal SSAT expression in luminal epithelial cells was higher (p < 0. 01) than that for glandular epithelial (GE) or stromal (ST) cells. Recombinant human insulin-like growth factor-I (IGF-I; 50 ng/ml) had no effect on steady-state SSAT mRNA levels, but it increased mitogenesis in all three cell types. In contrast, IGF-I caused a marked induction (p < 0.01) of SSAT mRNA levels in the human endometrial carcinoma cell line Hec-1-A. Uterine explants incubated with interleukin-6, transforming growth factor alpha, epidermal growth factor (each at 1, 10, and 100 ng/ml), retinoic acid and retinol (each at 0.01, 0.1, and 1 microM), and estradiol-17beta (10 nM) had SSAT mRNA levels similar to controls. By contrast, leukemia inhibitory factor (LIF; at 10 and 100 ng/ml) caused a modest, but significant (p < 0.05), increase in SSAT mRNA levels over those of untreated explants. This effect of LIF, however, did not approach the level of induction observed in GE or ST cells after addition of medium conditioned by Day 12 or 17 porcine conceptuses and in endometrial explants supplemented with medium conditioned by Day 21 porcine conceptuses or a continuous cell line (Jag-1) derived from Day 14 porcine trophoblast. We suggest that transient induction of endometrial SSAT gene expression at implantation is mediated by the functional interactions of specific conceptus-derived regulatory factors, distinct from estrogen, with endometrial-derived factor(s) such as LIF. These complex interactions are probably requisite for the transient, yet dramatic, induction of SSAT gene expression and may be critical for successful implantation.

Acetyltransferases↗

PubMed1998

A distal regulatory region of the insulin-like growth factor binding protein-2 (IGFBP-2) gene interacts with the basic helix-loop-helix transcription factor, AP-4.

Insulin-like growth factor binding protein-2 (IGFBP-2), the predominant IGFBP in the fetal circulation and an induced protein during several types of malignancies, belongs to a family of structurally related proteins that bind the mitogens, IGF-1 and IGF-2. The present study focused on functional analysis of the 5 '-flanking region (approximately 1.3 kb) of the IGFBP-2 gene to identify nuclear factors that mediate hepatic transcription of this gene. Luciferase (LUC) reporter constructs containing progressive deletions of 5'-flanking DNA and the intact promoter of the porcine IGFBP-2 gene were examined for functional activity by transient transfection of human HepG2 liver cells. LUC activity of the transfected reporter gene driven by the IGFBP-2 promoter and flanking sequences to -1397 (numbering relative to initiation codon at +1) was 22-fold higher than that of promoterless parent LUC vector. This activity was decreased by 60% with deletion of sequences to -874 bp, and dropped to basal levels with further truncation to -764 bp. The region between -874 and -765 bp (110 bp) functioned as a potent stimulator of heterologous SV40 promoter activity (110 bp/SV40-LUC construct) and was found to contain two noncontiguous basic helix-loop-helix (bHLH) transcription factor binding motifs (E-boxes [CAN NTG]: CACCTG and CAAATG). In electrophoretic mobility shift assays, nuclear proteins prepared from HepG2 cells formed two complexes (C1, C2) with double-stranded oligonucleotides containing either HLH sequence, mutations of which resulted in loss of complex formation. Southwestern blot analysis identified an HepG2 nuclear protein with molecular mass of 48 kDa, similar to that of the bHLH transcription factor AP-4, which bound the CACCTG motif. Cotransfection of HepG2 cells with the 110-bp/SV40-LUC construct and an expression vector encoding human AP-4 increased IGFBP-2 fragment-dependent SV40 promoter activity by 16-fold. This AP-4-mediated stimulation was lost following block mutation of both bHLH motifs within the IGFBP-2 110-bp fragment. Results demonstrate the functional importance of sequences upstream of the promoter in IGFBP-2 gene transcription and identify a novel mechanism by which bHLH proteins potentially may affect cell proliferation and differentiation via induction of IGFBP-2 synthesis.

DNA-Binding Proteins↗

PubMed1998

Proteolysis of insulin-like growth factor-binding proteins (IGFBPs) within the pig uterine lumen associated with peri-implantation conceptus development.

Pig conceptuses undergo morphological development from spherical to filamentous forms during days 10 to 12 of pregnancy, coincident with a high content of mRNAs encoding insulin-like growth factor (IGF)-I in the uterine endometrium and secretion of IGF-I into the uterine lumen. The potential regulation by developing conceptuses of the bioavailability of IGF-binding proteins (IGFBPs) within the uterine microenvironment was investigated. Uterine luminal flushings (ULFs) were obtained between days 10 and 18 of pregnancy and the presence of specific IGFBPs was detected by ligand blot analysis. ULFs collected at days 10 and 11 of pregnancy contained 46 and 43 kDa IGFBP-3, several IGFBPs of about 30 kDa including IGFBP-2, and an unidentified 26 kDa IGFBP; IGFBP-3 was the most abundant. By day 12, however, IGFBPs were substantially diminished or undetectable. Examination of the morphology of flushed conceptuses revealed that the loss of IGFBPs in ULF was associated with the transition from spherical to filamentous morphology. The abundance of IGFBP-3 mRNA in uterine endometrium, as monitored by blot-hybridization, was not altered in a similar way, suggesting that lack of IGFBP-3 in 'filamentous' ULF resulted from proteolysis rather than from decreased expression of the IGFBP-3 gene. Consistent with this, incubation of 'spherical' ULF with or without added 'filamentous' ULF at 37 degrees C resulted in the disappearance of endogenous IGFBP-3 only in 'spherical + filamentous' ULF. The protease activity in 'filamentous' ULF was inhibited by EDTA, but unlike matrix metalloproteinases, was not zinc ion-dependent or inhibited by 1,10-phenanthroline. Moreover, this activity was partially inhibited by the serine protease inhibitor aprotinin, but not by 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), a known inhibitor of plasmin. The IGFBP protease activity of ULF may therefore comprise a group of enzymes including an unidentified serine protease. The results suggest that elongating pig conceptuses induce IGFBP protease activity which may increase the intrauterine bioavailability of IGF.

Animals↗

PubMed1998

Relationship between blood serum insulin-like growth factor I concentration and postweaning feed efficiency of crossbred cattle at three levels of dietary intake.

Blood serum concentration of IGF-I was analyzed to determine its relationship with individual postweaning feed efficiency (gain/feed) of 36 crossbred steer calves fed at three levels of feed intake (n = 12 at each level). Diets consisted of a corn silage-based growing diet for 84 d followed by a 91% concentrate finishing diet for 56 d. Dietary intake levels were at 80, 90, or 100% of ad libitum. Diets were formulated to ensure equal daily intake of protein, vitamins, and minerals across intake treatment levels. Intake was measured daily; ADG, DMI, and feed efficiency were calculated at 28-d intervals, through d 140. Individual weights and serum samples were collected at the beginning of the study and at 28-d intervals thereafter. The IGF-I concentrations were determined with a RIA. Data were analyzed as a multivariate split-plot in time. Imposed dietary intake restrictions did not affect serum IGF-I concentration (P = .90) or individual feed efficiency (P = .36), even though the least squares means for IGF-I concentration tended to decrease and the feed efficiency means tended to increase under the restricted intake levels. Serum IGF-I concentration, ADG, and feed efficiency were affected (P < .001) by collection date. Residual correlations between IGF-I concentrations at adjacent 28-d sampling times averaged .72. Diet intake level x sampling time interactions existed for ADG (P = .02) and feed efficiency (P < .001). Positive residual correlations of .28 (P < .001) and .16 (P = .07) existed between IGF-I and ADG and between IGF-I and feed efficiency, respectively. Regression analysis indicated that a 1 ng/mL increase in serum IGF-I concentration was associated with a .00135 kg/d increase in ADG (P < .001) and a .0001 kg gain/kg feed increase in feed efficiency (P = .04). These results support the hypothesis that serum IGF-I plays a role in growth and in efficiency of feed utilization in beef cattle.

Animal Feed↗