Showing papers in "Molecular Endocrinology in 1989"
TL;DR: Findings suggest that although IGF-I is not produced by breast epithelial cells it may function as either a paracrine stimulator of epithel cells or an autocrinestimulator of stromal cells.
Abstract: Insulin-like growth factor I (IGF-I) activity has been reported to be produced by several human cancers Identification of RNAs transcribed from the IGF-I gene has been complicated by the detection of multiple hybridizing bands on Northern analysis To determine if any of these RNAs are transcribed from the IGF-I gene, we have used a sensitive and specific ribonuclease (RNAse) protection assay for IGF-I We have also studied the breast cancer tissue expression of IGF-I using in situ hybridization histochemistry We have found no IGF-I mRNA in breast (zero of 11) or colon cancer (zero of 9) cell lines; both of these tumors have been previously reported to express IGF-I mRNA However, three of three neuroepithelioma and one of two Ewing's sarcoma cell lines express IGF-I mRNA; therefore, in these tumors IGF-I may be an autocrine growth factor In contrast to breast cancer cell lines, RNA extracted from breast tissues has easily detectable IGF-I mRNA In situ hybridizations show that IGF-I mRNA is expressed
420 citations
TL;DR: Androgen administration to 4-day castrated rats led to a marked decrease in TGF beta mRNA to a level comparable to its constitutive expression obtained in the intact control animals, indicating that expression of TGFbeta in the rat ventral prostate is under negative androgenic regulation.
Abstract: Castration-induced androgen deprivation leads to the activation of the programmed death of the androgen-dependent prostatic epithelial cells in the rat ventral prostate. In order to identify potential mediators of this programmed cell death, the expression of transforming growth factor-beta (TGF beta) in the rat ventral prostate was studied, after castration induced-androgen withdrawal. Steady state levels of TGF beta mRNA were determined by Northern blot analysis and compared with mRNA levels for prostatein C3, the major androgen-dependent secretory protein of ventral prostate and also with mRNA levels for TRPM-2, a gene that is specifically expressed during castration induced prostatic cell death. Within the first day after castration there was a dramatic increase in the levels of TGF beta mRNA in the ventral prostate (approximately 10-fold) and by 4 days after castration TGF beta mRNA was maximally expressed (approximately 40-fold increase), by which time the androgen-dependent C3 secretory protein mRNA transcripts have diminished to undetectable levels. Androgen administration to 4-day castrated rats led to a marked decrease in TGF beta mRNA to a level comparable to its constitutive expression obtained in the intact control animals, indicating that expression of TGF beta in the rat ventral prostate is under negative androgenic regulation. The transcript levels encoding TRPM-2 initially increased 10-fold within the first day after castration and by day 4 post castration there was a dramatic increase (approximately 50-fold) which correlated well with the maximal rate of cell death of the androgen-dependent prostatic epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)
402 citations
TL;DR: The data indicate that steroid receptor mRNAs are differentially distributed in hippocampus, and that sensitivity to steroids occurs within defined structural domains of the hippocampal formation.
Abstract: Messenger RNAs coding for glucocorticoid (GR) and mineralocorticoid (MR) receptor proteins were localized to discrete subfields of the hippocampal formation by in situ hybridization histochemistry, using cRNA probes of approximately equivalent specific activity. Both GR and MR mRNAs were present in all subfields examined; GR mRNA was of greatest abundance in CA1, while MR mRNA was most densely labeled in CA3. In all subfields examined, MR mRNA was considerably more abundant than GR mRNA. Removal of circulating glucocorticoids by adrenalectomy precipitated an up-regulation of GR mRNA in subfields CA1-2 and the dentate gyrus, which was reversed by dexamethasone replacement. High doses of dexamethasone significantly down-regulated GR mRNA in CA3. In contrast, adrenalectomy produced significant up-regulation of MR mRNA only in subfield CA1-2. The data indicate that steroid receptor mRNAs are differentially distributed in hippocampus, and that sensitivity to steroids occurs within defined structural domains of the hippocampal formation.
313 citations
TL;DR: Although the functional significance of alternative splicing of the insulin receptor mRNA is unknown, differential expression of these two receptor mRNAs may provide a structural basis for previously observed tissue-specific differences in insulin binding and action.
Abstract: Two previously reported insulin receptor cDNA sequences differ by 36 base pairs (bp) in the distal alpha-subunit, suggesting that alternative mRNA splicing within the coding region may occur (two insulin receptor isoforms). We developed a quantitative modification of the polymerase chain reaction technique in order to detect and characterize differential mRNA splicing at this site within the distal alpha-subunit. Using RNA derived from a variety of human cell types, we detected two polymerase chain reaction-amplified cDNA species reflecting the presence or absence of the above 36 nucleotides. Identity of the two cDNA species was confirmed by Southern blots, the use of a BANI restriction site present only in the 36 base pair segment and dideoxy sequencing. The relative expression of the two mRNA forms varied markedly in a tissue-specific manner. Buffy coat leukocytes and Epstein-Barr virus-transformed lymphocytes express only the shorter mRNA. Placenta expresses both species equally; muscle, isolated adipocytes and cultured fibroblasts express somewhat more of the longer mRNA (relative ratios of mRNA abundance of 1.51, 3.18, and 2.77, respectively); liver expresses mostly the longer mRNA (relative ratio of 9.8). In RNA derived from cultured and fresh cells from patients with several states of insulin resistance, the relative expression of the two mRNA species was similar to results obtained with comparable normal tissues. Although the functional significance of alternative splicing of the insulin receptor mRNA is unknown, differential expression of these two receptor mRNAs may provide a structural basis for previously observed tissue-specific differences in insulin binding and action.
309 citations
TL;DR: Polyclonal antibodies raised against 3β-hydroxysteroid dehydrogenase isolated from human placenta were used to screen a λgt11 expression cDNA library from the same tissue, deducing the protein deduced from cDNA sequences contains 372 amino acids.
Abstract: Polyclonal antibodies raised against 3β-hydroxysteroid dehydrogenase isolated from human placenta were used to screen a λgt11 expression cDNA library from the same tissue. The protein deduced from cDNA sequences contains 372 amino acids with a calculated mol wt of 42,216. Since 3β-hydroxysteroid dehydrogenase is the enzyme catalyzing the formation of all classes of hormonal steroids, the availability of the cDNA encoding this enzyme opens new possibilities for a detailed investigation of the factors regulating the expression and activity of this crucial enzyme in adrenal, gonadal as well as peripheral tissues. INTRODUCTION The formation of Δ4-3-keto steroids from Δ5-ene, 3β-hydroxy precursors is a key step in the biosynthesis of all classes of hormonal steroids, namely glucocorticoids, mineralocorticoids, progesterone, androgens, and estrogens. This reaction is catalyzed by the enzymatic system 3β-hydroxy-5-ene-steroid dehydrogenase (EC 1.1.1.145) and steroid Δ5-Δ4-ene-isomerase (EC-5.3.3.1), hereafter ca...
309 citations
TL;DR: In this paper, two types of cDNA clones were isolated from cDNAs for the mouse GH receptor and the related serum GH binding protein, and it was speculated that these two types encoded the high and low molecular weight variants of the GH receptor/serum binding proteins, respectively.
Abstract: Predicted amino acid sequences for the mouse GH receptor and the related serum GH binding protein were deducted from cDNAs. Two types of cDNA clones were isolated. Both types coded an identical peptide domain with extensive homology to the extracellular domains of the recently cloned human and rabbit GH receptors. However, while one type of clone also encoded regions with homology to the transmembrane and cytoplasmic domains of the human and rabbit GH receptors, the other encoded a short hydrophilic carboxyl-terminal region in place of the transmembrane domain. It is speculated that these two types of clones encode the high and low molecular weight variants of the mouse GH receptor/serum binding proteins, respectively. The low molecular weight variant has been previously found to constitute the majority of the serum GH binding activity in mice. It is proposed that the substitution of the hydrophilic tail for the transmembrane domain may give the low molecular weight variant its soluble nature and account ...
308 citations
TL;DR: Human PRL receptor cDNA clones from hepatoma and breast cancer libraries were isolated by using a rat PRL receptors cDNA probe to better understand the role of PRL in the development and growth of human breast cancer.
Abstract: Human PRL receptor cDNA clones from hepatoma (Hep G2) and breast cancer (T-47D) libraries were isolated by using a rat PRL receptor cDNA probe. The nucleotide sequence predicts a mature protein of 598 amino acids with a much longer cytoplasmic domain than the rat liver PRL receptor. Although this extended region has additional segments of localized sequence identity with the human GH receptor, there is no identity with any consensus sequences known to be involved in hormonal signal transduction. This cDNA will be a valuable tool to better understand the role of PRL in the development and growth of human breast cancer.
280 citations
TL;DR: Since E2DH is the enzyme required for the formation of 17 beta-estradiol, the availability of the cDNA encoding the enzyme should permit a detailed investigation of the factors regulating the expression and activity of this crucial enzyme, in both normal and malignant tissues, especially breast cancer.
Abstract: Human placental estradiol 17β-dehydrogenase (E2DH) cDNA clones were isolated from a λgt11 expression library by screening with 33 mer synthetic oligonucleotides derived from the amino acid sequence of the catalytic site of E2DH and with polyclonal antibodies raised against the enzyme purified from human placenta. Using 32P-labeled fragments from the coding and 5′-untranslated regions, two mRNA species have been identified in poly(A)+ RNA from human placenta, a major species migrating at 1.3 kilobases (kb) while a minor one is found at 2.2 kb. Primer extension analysis identifies the major mRNA as starting 9–10 nucleotides upstream from the in-frame ATG initiating codon while the longer mRNA has at least 814 noncoding nucleotides at its 5′-terminus. Sequence analysis of the longest cDNA clone (2092 base pairs) shows that this clone possesses identical coding and noncoding sequences in the regions of overlap with the shorter cDNA clones. The 32P-labeled 5′-noncoding fragment hybridized only to the 2.2 kb ba...
274 citations
TL;DR: Analysis of the recombinant expression in mammalian cells and the purification to apparent homogeneity of human TGFβ3 revealed that it exerts activities similar to the two other TGF β species, but that there are distinct differences in potencies between the different T GFβ forms depending on the cell type and assay used.
Abstract: Recent cDNA characterization has predicted the existence of a new member of the transforming growth factor family, transforming growth factor-beta 3 (TGF beta 3). However, nothing is known about the biological activities of the TGF beta 3 protein, since it has not been purified from any natural sources. We report here the recombinant expression in mammalian cells and the purification to apparent homogeneity of human TGF beta 3. The TGF beta 3 was evaluated in comparison with purified TGF beta 1 and TGF beta 2 in several assays for its effects on stimulation or inhibition of proliferation of mammalian cells. These analyses revealed that TGF beta 3 exerts activities similar to the two other TGF beta species, but that there are distinct differences in potencies between the different TGF beta forms depending on the cell type and assay used.
274 citations
TL;DR: It is demonstrated that the chick c-erbA-alpha possesses hormone-independent (constitutive) activity in cells which express endogenous rat thyroid hormone receptor, and that this activity is abolished upon deletion of the conserved heptad repeats.
Abstract: The thyroid hormones and retinoic acid are potent modulators of differentiation, development, and gene expression. The transcriptional activities of these ligands are mediated by closely related nuclear receptors which bind and activate identical hormone responsive DNA elements. We noticed that a region within the ligand binding or E domain is well conserved between receptors for these hormones. This region contains hydrophobic heptad repeats that are structurally similar to the leucine-zipper dimerization domain. To study the function of this conserved domain, we examined the transcriptional responses of thyroid hormone receptor/c-erbA deletion mutants which lacked the heptad repeats. We previously reported that the chick c-erbA-alpha possesses hormone-independent (constitutive) activity in cells which express endogenous rat thyroid hormone receptor. We now demonstrate that this activity is abolished upon deletion of the conserved heptad repeats. This suggests that the heptad repeats mediate in vivo interactions between chick c-erbA and rat thyroid hormone receptors. To further test this hypothesis deletion mutants of chick c-erbA were constructed which contained all eight heptad repeats but which lacked the zinc-finger DNA binding domain. Although these mutants are transcriptionally inactive, they act in a dominant-negative fashion to block trans-activation by both the chick c-erbA-alpha and the endogenous thyroid hormone and retinoic acid receptors. We suggest that the heptad repeats mediate the formation of inactive mutant/wild-type hetero-dimers. Dimer formation suggests a mechanism to account for the dominant-negative phenotypes displayed by nonhormone binding variants of c-erbA, the proto-oncoprotein v-erbA and patients with the generalized thyroid hormone resistance syndrome.
274 citations
TL;DR: Analysis of the effects of estrogen and progestin, their antagonists, and growth factors on the regulation of estrogen receptor (ER) mRNA and protein levels in T47D breast cancer cells, and in two sublines of MCF-7 cells which contain high ER levels found the effect of E2 on ER levels was dependent on the prior growth history of the cells.
Abstract: Since sex steroid hormones and growth factors are known to modulate the proliferation of breast tumors, we have studied the effects of estrogen and progestin, their antagonists, and growth factors on the regulation of estrogen receptor (ER) mRNA and protein levels in T47D breast cancer cells, which contain low levels of ER, and in two sublines of MCF-7 cells which contain high ER levels. The mRNA levels were measured by Northern blot analysis using lambda OR8, a cDNA probe for ER, and protein levels were measured by hormone binding or Western blot analysis. Treatment of T47D cells with estradiol (E2) caused a 2.5-fold increase in ER mRNA (6.6 kilobases) levels after 48 h. The progestin R5020 evoked a marked decrease in ER mRNA and protein levels to 20% of control values, while the antiprogestin RU38,486 caused no change in ER. In MCF-7 cells, the effect of E2 on ER levels was dependent on the prior growth history of the cells. In cells grown in low estrogen [5% charcoal-dextran-treated calf serum with phenol red for 8 yr (MCF-7-K2)], which are still E2 responsive, treatment with E2, the antiestrogen LY117018, or both produced little change in ER mRNA or protein; in contrast, ER mRNA and protein were reduced by E2 to 40% and 50% of control levels, respectively, in MCF-7 cells (denoted MCF-7-K1) which had been maintained routinely in medium containing 5% calf serum. This decrease in ER mRNA was dose dependent; 10(-11) E2 reduced levels to 60%, and 10(-10) M E2 evoked the maximal drop to 40% of the control level in 2 days. LY117018 alone did not alter ER mRNA levels in these cells, but it completely prevented the down-regulation of ER by E2. Administration of progestin, but not antiprogestin, along with E2 partially prevented the decrease in ER evoked by E2. Addition of epidermal growth factor or insulin-like growth factor-I to MCF-7-K1 cells, which increased cell proliferation, had no detectable effect on ER levels. Treatment with transforming growth factor-beta, which decreased cell proliferation, reduced ER by about 20%.(ABSTRACT TRUNCATED AT 400 WORDS)
TL;DR: Insulin-like growth factor-II was slightly less potent than IGF-I in stimulating DNA synthesis in MCF-71 cells, an effect that paralleled its ability to compete for [125I]IGF-I binding in these cells.
Abstract: Insulin-like growth factor-II (IGF-II) is a potent mitogen for several types of cultured cells and tissues. We have studied the interaction of IGF-II with a panel of cultured human breast cancer cell lines, examining the possibility that these cells synthesize and secrete IGF-II activity which could have autocrine/paracrine functions. Synthetic IGF-II was mitogenic in five of seven cell lines tested, including the estrogen receptor-positive lines MCF-7L, ZR75-1, and T47D and the estrogen receptor (ER)-negative lines Hs578T and MDA-231. IGF-II was slightly less potent than IGF-I in stimulating DNA synthesis in MCF-71 cells, an effect that paralleled its ability to compete for [125I]IGF-I binding in these cells. Affinity labeling studies revealed that IGF-II could also compete for binding to the 130,000 mol wt alpha-subunit of the IGF-I receptor. A monoclonal antibody to the IGF-I receptor inhibited the mitogenic effects of IGF-II in MCF-7L and MDA-231 cells, suggesting that this receptor mediates the growth effects of IGF-II in these breast cancer cells. Using a RIA and a RRA, IGF-II-like activity was detected in conditioned medium extracts processed to remove IGF-binding proteins from several breast cancer cell lines, with the highest levels found in conditioned medium from MCF-7L and T47D cell lines. IGF-II mRNA transcripts in MCF-7L and T47D cells were identified by Northern blot analysis and were confirmed by RNase protection assay. IGF-II mRNA was increased by estrogen in MCF-7L cells.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: Primary pituitary cell cultures derived from adult male rats were used to explore the direct effects of purified porcine inhibin and follistatin, and recombinant human activin A on FSH beta, as well as LH beta and alpha-subunit mRNA levels, to demonstrate that the actions of these gonadal peptides on F SH secretion may be accounted for, at least in part at the level of biosynthesis.
Abstract: Primary pituitary cell cultures derived from adult male rats were used to explore the direct effects of purified porcine inhibin and follistatin, and recombinant human activin A on FSHβ, as well as LHβ and α-subunit mRNA levels. Subunit mRNAs were determined by blot hybridization using α, LHβ, and FSHβ cDNA and genomic fragments. Treatment with inhibin for 72 h significantly suppressed α and FSHβ mRNA levels with parallel changes in FSH secretion. No change in LHβ mRNA levels was observed. A decrease in FSHβ mRNA to undetectable levels was seen 4 h after inhibin administration. Recombinant human Activin A caused dose-dependent and parallel increases in FSHβ mRNA levels and FSH secretion. This increase was evident at 4 h after activin administration and maintained at longer times, α and LHβ mRNA levels remained unchanged. Follistatin addition to cultures for 72 h significantly reduced FSHβ mRNA levels. In a time-course experiment, a reduction in FSHβ mRNA to undetectable levels was observed 24 h after foll...
TL;DR: The results suggest that the varied actions of PRL may involve multiple receptors that are part of distinct signal transduction pathways.
Abstract: We have characterized the PRL receptor (PRL-R) present in mouse liver by purification, cross-linking, and immunological analysis of the protein, and by the isolation of PRL-R cDNA clones. Analysis of the cDNA clones indicates that the liver receptor is actually a family of proteins. Two of these proteins are predicted to be synthesized as precursors of 303 and 292 amino acids, with common signal sequences, extracellular domains, and transmembrane domains; a portion of their cytoplasmic domains are also identical, but these proteins differ markedly in the terminal region of this domain. A third PRL-R protein is predicted to be a truncated form and may be secreted. These multiple PRL-R mRNAs appear to be encoded by at least two genes, with the sequence variation for the two full-length proteins likely due to alternative RNA splicing. These results suggest that the varied actions of PRL may involve multiple receptors that are part of distinct signal transduction pathways.
TL;DR: A consensus T3 receptor binding half-site, AGGT(C/A)A, at least two copies of which are required for a T3 response is proposed, showing that all three domains contribute to the amplified T2 response.
Abstract: We have previously identified sequences required for thyroid hormone (T3) induction of the rat GH (rGH) promoter, which lie in a region from -188 to -164 upstream of the mRNA start site. Within this region, Domains A, -189 to -184 and B, -179 to -174, are imperfect direct repeats, and domain C, -172 to -167, is a divergent inverted copy that matches the A domain at 4/6 positions. A series of synthetic mutant versions of this sequence were inserted upstream of a truncated rGH promoter, or as a replacement for wild-type sequences in a synthetic 237 base pair rGH promoter or upstream of the heterologous thymidine kinase promoter. Mutations changing the B domain to a perfect copy of the A domain significantly increased T3 induction (21.3-fold) relative to the wild type (3.6-fold). A single point mutation making the C domain a better match to the A domain also increased T3 induction to 16.2-fold. Combining this up-mutation with any of three down-mutations in the A, B, or C domains strongly decreased response, showing that all three domains contribute to the amplified T3 response. Binding affinity of the various mutant oligonucleotides was assessed using in vitro translated receptor and affinity paralleled the functional responses for most binding site mutations. Requirements for in vitro binding were, however, less rigorous than those for functional T3 induction. Based on these results, we propose a consensus T3 receptor binding half-site, AGGT(C/A)A, at least two copies of which are required for a T3 response.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: The patterns of expression suggest a physiological role for TGF-beta 2 both in embryonic development and in the maintenance of adult tissues.
Abstract: Murine transforming growth factor-β2 (TGF-β2) cDNAs were isolated from cDNA libraries derived from a differentiated murine embryonic carcinoma cell line, PCC3. The composite cDNA sequence is 4267 nucleotides long, including a 1217 nucleotides 5′-untranslated sequence, and encodes a murine TGF-β2 precursor of 414 amino acids with 96% identity to its human counterpart. Several consensus polyadenylation sequences are present in the 1807 nucleotides 3′-untranslated sequence. Five TGF-β2 mRNA species are observed in the developing mouse fetus and they show different patterns of expression during development. TGF-β2 mRNA expression was also examined in adult mouse tissues, in which four of the five RNA species were observed. TGF-β2 mRNAs were present in all adult mouse tissues examined, except liver, and was most abundant in placenta, the male submaxillary gland and lung. The patterns of expression suggest a physiological role for TGF-β2 both in embryonic development and in the maintenance of adult tissues.
TL;DR: It is suggested that nuclear factors interact with the T3 receptor to enhance hormone-independent binding to a TRE and form two receptor-DNA complexes for both the alpha- and beta-receptors.
Abstract: Using a gel shift assay, we analyzed the binding of in vitro translated α- and β-thyroid hormone (T3) receptors to a T3-response element (TRE) derived from the rat GH gene. No receptor-TRE complexes were observed when translated receptor alone was incubated with the TRE. However, addition of a nuclear extract from liver to the translational products resulted in the formation of two receptor-DNA complexes for both the α- and β-receptors. These complexes were shown to contain translated receptor by comigration of 32P-labeled TRE and 35S-labeled receptor in the gel shift assay. A competition experiment demonstrated that formation of the complexes was sequence specific. Preincubation of the liver nuclear extract at 60 C abolished formation of both complexes indicating that receptor-TRE binding required a heat-labile nuclear factor. Phosphocellulose chromatography of the nuclear extract resulted in separation of the activities required for formation of the two complexes. Analysis of nuclear extracts from diffe...
TL;DR: The E-induced increase in P-responsive neurons in ventromedial hypothalamus can account for the permissive influence of E on P-facilitated reproductive behavior, the first demonstration that synthesis of a transcription factor (PR) can be related to a mammalian behavior.
Abstract: Diverse effects of steroid hormones on different tissues result from the tissue-specific regulation of target gene expression by steroid hormone receptors. These receptors belong to a family of transacting factors that regulate transcriptional activation of target genes by binding to DNA recognition sequences located in the 5'-flanking region of the target gene. In the brain, receptors for the gonadal steroid hormones estrogen (E) and progesterone (P) are present in discrete neuronal populations. These steroid hormone receptor-containing neurons mediate the effects of the gonadal steroids on a number of neural processes, including reproductive behavior. Using in situ hybridization we have found progesterone receptor (PR) mRNA-containing neurons present in specific hypothalamic nuclei and in the amygdala. E regulates PR mRNA levels in specific neuronal cell groups which express both ER and PR (in basomedial hypothalamus), but not in others (medial amygdala). The E-induced increase in P-responsive neurons in ventromedial hypothalamus can account for the permissive influence of E on P-facilitated reproductive behavior. This is the first demonstration that synthesis of a transcription factor (PR) can be related to a mammalian behavior.
TL;DR: The results demonstrate that the primary structure and regulated expression of IGF-I by GH have been conserved in teleosts.
Abstract: Protein and cDNA sequence analysis have revealed that the insulin-like growth factor (IGF-I) has been highly conserved among several mammalian species. Using the combined techniques of polymerase chain reaction and molecular cloning, we have now obtained the cDNA sequence encoding preprolGF-I from a teleost species, Oncorhynchus kisutch (coho salmon). The 2020 nucleotide (nt) cloned cDNA sequence contains a 528 nt open reading frame encoding 176 amino acids in preprolGF-I and 175 nt and 1317 nt of flanking 5′- and 3′-untranslated regions, respectively. The deduced amino acid sequence of salmon IGF-I is highly conserved relative to its mammalian homologues and there are only 14 amino acid differences out of 70 between salmon and human IGF-I. Interestingly, the C-terminal E domain of salmon prolGF-I, which is presumed to be proteolytically cleaved during biosynthesis, also shows striking amino acid sequence homology with its mammalian counterpart, except for an internal 27 residue segment that is unique to ...
TL;DR: The broad tissue distribution of follistsatin mRNA strongly suggests other physiological roles for follistatin besides the inhibition of pituitary FSH release and highly homologous to porcine and human follistatins.
Abstract: Follistatin is a glycosylated single-chain protein originally isolated from porcine follicular fluid. It specifically inhibits the secretion of FSH from the pituitary. We have now isolated and characterized a cDNA for rat follistatin from the PMSG-stimulated ovarian library. The deduced amino acid sequence of the rat follistatin precursor is highly homologous (greater than 98%) to porcine and human follistatins including potential Asn-glycosylation sites. The genomic clone encoding rat follistatin was also isolated and revealed that the exon and intron organization of the follistatin gene structure is conserved among rat, porcine, and human. Northern analyses in rat tissues demonstrated that the follistatin gene is expressed not only in the ovary but also in the kidney and brain. In the immature rat ovary, the follistatin mRNA level is stimulated by PMSG injection (20 IU/rat), but is not affected by human CG (10 IU/rat) after PMSG administration. In situ hybridization studies revealed that the mRNA level in the ovary was low in primordial follicles, but dramatically increased in the granulosa cells of the growing secondary and tertiary follicles and then decreased in the mature preovulatory follicles. A strong follistatin mRNA signal was observed over the collecting tubules of the outer medulla of the kidney, and a weak to moderate signal was detected in brain. The broad tissue distribution of follistatin mRNA strongly suggests other physiological roles for follistatin besides the inhibition of pituitary FSH release.
TL;DR: Using 32P-labeled cRNA probes directed against Type I and Type II receptor mRNA to screen various tissues, and investigating the effect of adrenalectomy and dexamethasone administration on their levels in hippocampus found Type I mRNA to be high in hippocampus, colon, and heart; low in liver; and undetectable in thymus.
Abstract: We have used 32P-labeled cRNA probes directed against Type I (mineralocorticoid, high affinity glucocorticoid) and Type II (classical glucocorticoid) receptor mRNA to screen various tissues, and have investigated the effect of adrenalectomy (ADX) and dexamethasone (DM) administration on their levels in hippocampus. Both Northern blot and S1 nuclease analysis showed Type I mRNA to be high in hippocampus, colon, and heart; low in liver; and undetectable in thymus. Type II mRNA was high in liver, thymus, and brain; and low in testis and parotid. A transient increase in both hippocampal Type I and Type II mRNA was noted at 1–3 days post ADX. DM similarly elicited a rise in hippocampal Type I mRNA at 2–4 days after ADX, but prevented the ADX-induced increment in Type II mRNA. In contrast to the transient increase in Type I receptor mRNA levels, hippocampal levels of Type I receptors measured by [3H]aldosterone binding were constant 1–16 days post ADX. DM administration caused a doubling in Type I receptor leve...
TL;DR: The results demonstrate that cathepsin-D and pS2 genes are under complex regulation in MCF7 cells, since growth factors stimulate their expression via indirect mechanisms contrasting with the primary transcriptional effects of estrogens.
Abstract: In MCF7 human breast cancer cells, cathepsin-D and pS2 mRNAs are specifically and directly induced by estrogens at the transcriptional level We studied the regulation of expression of these two genes by growth factors that are also mitogenic in this cell line We show that pS2 mRNA, like cathepsin-D mRNA, is rapidly induced 2- to 4-fold by epidermal growth factor The effect of epidermal growth factor on these two mRNAs was dependent upon de novo protein synthesis, indicating a different mechanism of regulation than with estradiol Other peptide growth factors, such as insulin, insulin-like growth factor I, and basic fibroblast growth factor, also increased up to 3-fold the steady state levels of the two mRNAs in MCF7 cells The pS2 mRNA, but not cathepsin-D mRNA, was also induced up to 8-fold by protein kinase-C activation with 12-O-tetradecanoylphorbol-13-acetate, suggesting the possible involvement of this transduction pathway in pS2 mRNA induction The effect of 12-O-tetradecanoylphorbol-13-acetate w
TL;DR: The estrogen receptor (ER) is present in a wide variety of mammalian tissues and is required for physiological estrogen responses, including estrogen-induced tissue-specific changes in gene expression, but the ER mRNA regulation by E2 differed in direction depending on the tissue examined.
Abstract: The estrogen receptor (ER) is present in a wide variety of mammalian tissues and is required for physiological estrogen responses, including estrogen-induced tissue-specific changes in gene expression. We studied the estrogen regulation of the mRNAs encoding the ER in rat uterus, liver, and pituitary. Ovariectomized (21-28 day post surgery) female CD-1 rats were injected daily with 17 beta-estradiol (E2, 10 micrograms/100 g BW) for 0, 1, or 4 h, 1, 3, or 7 days and compared with intact controls. Steady-state levels of ER mRNA were quantified using a human ER cDNA probe. Only one hybridizing species of approximately 6.2 kilobase (kb) was detected in uterine and liver RNA, similar to that observed in MCF7 human breast cancer cells. However, the ER mRNA regulation by E2 differed in direction depending on the tissue examined. In uterus, ER mRNA increased 3- to 6-fold after ovariectomy, and returned to intact levels within 24 h of E2 replacement. In contrast, liver ER mRNA declined 1.5- to 3-fold after ovariectomy and returned to intact levels after 1-3 days of E2. In pituitary tissue two hybridizing forms of ER mRNA were observed, with one species migrating at 6.2 kb, equivalent to the form in other tissues, and a second smaller species at approximately 5.5 kb. The lower molecular weight species varied somewhat in abundance from animal to animal, averaging about 20% of the intensity of the 6.2 kb band. The ER mRNA forms were regulated positively with E2.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: This report constitutes the first identification of ER mRNA from a fish and shows two domains of striking amino acid homology with human, avian, and Xenopus estrogen receptors (ERs) with cDNA binding region 90%, Hormone binding region 60%.
Abstract: The estrogen-binding region of the cDNA for chicken ER reveals a mRNA of 3.5 kilobases (kb) in rainbow trout liver. The level of this messenger, which is very low in the liver of naive male animals, can be increased by estrogen stimulation. With this chicken probe, we have isolated a clone from a λ gt10 trout liver cDNA library. The partial cDNA sequence, which encompasses most of the coding region, shows two domains of striking amino acid homology with human, avian, and Xenopus estrogen receptors (ERs) (DNA binding region: 90%, Hormone binding region: 60%). With this specific probe rainbow trout ER, we detected another messenger (4.5 kb) that is less expressed than the 3.5 kb messenger. The kinetics of stimulation of the two messengers is compared with the kinetics of accumulation of vitellogenin mRNA after E2 administration. This report constitutes the first identification of ER mRNA from a fish.
TL;DR: In this paper, a series of recombinant cDNA clones corresponding to the mRNA for bTP-1 have been isolated from cDNA libraries representing day 18−19 bovine conceptus poly(A)+ mRNA.
Abstract: Bovine trophoblast protein-1 (bTP-1) is a secreted glycoprotein that consists of several forms differing slightly in mol wt and isoelectric point. It is produced by bovine conceptuses after about day 15 of pregnancy and is believed to play a key role in signalling the presence of an embryo to the mother. In this study, a series of recombinant cDNA clones corresponding to the mRNA for bTP-1 have been isolated from cDNA libraries representing day 18–19 bovine conceptus poly(A)+ mRNA. Base sequencing of several cDNAs indicated that multiple mRNAs for bTP-1 exist. Northern blotting and primer extension experiments showed that the mRNAs average about 1 kilobase in length. One apparently full-length cDNA clone consisted of 1035 bases up to the beginning of the poly(A) tail. It contained an open reading frame of 195 codons which began at a position 79 bases from the 5′ end. Its entire sequence was 85% identical to that of a cDNA for the immunologically related ovine trophoblast protein-1 (oTP-1) and about 79% id...
TL;DR: In AtT-20 and rat pituitary cells, processing of POMC through at least two endo- and exoproteolytic cleavage steps and alpha-amidation of joining peptide begin in the trans Golgi subcompartment, it is concluded that this Golgi cisterna as well as secretion granules contain the active enzymes necessary for proteolytic processing and alpha -amidation.
Abstract: The intracellular sites where proteolytic processing of pro-ACTH/endorphin or POMC take place have not yet been reliably identified. We have used affinity-purified antisera that recognize only the products of POMC processing and immunoelectron microscopy to identify the compartments of rat pituitary corticotropes and mouse AtT-20 cells in which cleavage occurs. Immunoperoxidase labeling of cryostat sections and immunogold labeling of ultrathin frozen sections were used for localization of the processing sites. By both procedures we detected processed peptides in Golgi cisternae and secretion granules. Within the Golgi, labeling was limited to the last or transmost cisterna and was most concentrated in its dilated rims which contain condensing secretory protein. No labeling of other Golgi cisternae was seen. All Golgi cisternae were labeled, however, when antisera that recognize unprocessed POMC were used for immunolabeling. We conclude that in AtT-20 and rat pituitary cells: 1) processing of POMC through ...
TL;DR: Rat serum contains two major forms of insulin-like growth factor (IGF) binding proteins (BPs) that have apparent mol wts of about 35,000 and 150,000, and a cDNA clone encoding an IGF-BP whose N-terminal sequence is completely homologous to the NH2- terminal of the Buffalo rat liver cells-3A BP is isolated.
Abstract: Rat serum contains two major forms of insulin-like growth factor (IGF) binding proteins (BPs) that have apparent mol wts of about 35,000 and 150,000. We have isolated a cDNA clone encoding an IGF-BP whose N-terminal sequence is completely homologous to the NH2-terminal of the Buffalo rat liver cells-3A BP. The 270 amino acid mature protein has a predicted mol wt of 29,500. It contains a cysteine rich domain at each end of the molecule and an Arg-Gly-Asp (RGD) tripeptide motif near its C-terminus which suggests that this BP might associate with integrin cell surface receptors. The mature protein shares only partial homology with two published human IGF-BPs. Northern blot analysis shows that its mRNA is abundant in several fetal tissues, in adult brain, testes, ovaries, and kidney. Expression in the liver is high in fetal life but decreases to a barely detectable level in adulthood. However, upon hypophysectomy, the mRNA level increases at least 20-fold which suggests a hormonal regulation for the hepatic p...
TL;DR: Analysis indicated that binding to the high Mr proteoglycan is not necessary for TGF beta 1 induced regulation of DNA, collagen and fibronectin synthesis, change in cell morphology, or reorganization of the actin cytoskeleton, and it is proposed that the lower Mr components are the active receptors mediating these events.
Abstract: Transforming growth factor-β (TGFβ1 and TGFβ2) bind to several different cell surface proteins, including a high Mr proteoglycan. We found that on primary and early passage cultures of fibroblasts, chondroblasts, and osteoblasts TGFβ1 binds to both the high Mr proteoglycan and to lower Mr components, whereas on epithelial, endothelial, and lymphoid-derived cells TGFβ1 only binds to the lower Mr species. With cell lines, this distinction is lost. Further analysis indicated that binding to the high Mr proteoglycan is not necessary for TGFβ1 induced regulation of DNA, collagen and fibronectin synthesis, change in cell morphology, or reorganization of the actin cytoskeleton. We propose that the lower Mr components are the active receptors mediating these events
TL;DR: A cDNA clone encoding the complete rat 17α-hydroxylase (P45017α) from testis has been identified and sequenced and the deduced amino acid sequence is found to have 69% similarity with human P45017 α, 64% similarities with bovine P 45017α, and 47% similarityWith chicken P452017α.
Abstract: A cDNA clone encoding the complete rat 17α-hydroxylase (P45017α) from testis has been identified and sequenced. The deduced amino acid sequence is found to have 69% similarity with human P45017α, 64% similarity with bovine P45017α, and 47% similarity with chicken P45017α. The protein contains 507 amino acids being one amino acid shorter than the human P45017α as the result of a codon being absent at the position of amino acid 139 in the human sequence. The cDNA hybridizes to a single mRNA (∼2.0 kilobases) in rat testis RNA and Southern analysis indicates the presence of a single CYP17 gene in the rat genome. Expression of this cDNA in COS1 cells leads to production of a steroid hydroxylase which is capable of converting both 17α-hydroxypregnenolone and 17α-hydroxyprogesterone into C19 steroids, dehydroepiandrosterone, and androstenedione, respectively. This activity profile is distinct from that of either the human or bovine forms of P45017α which are unable to catalyze 17,20-lyase conversion of Δ4-C21 st...
TL;DR: Data is presented that indicates MDA-468 cells secrete a transforming growth factor with autocrine growth stimulatory capabilities.
Abstract: The response of malignant and nonmalignant human breast cell lines to the growth inhibitory effects of monoclonal antibodies against the epidermal growth factor (EGF) receptor was studied. A series of human breast cell lines, which express EGF receptor, were used: MDA-468, MDA-231, and Hs578T human breast cancer cells and the transformed human mammary epithelial cell lines 184A1N4 and 184A1N4-T that have been benzo[a]pyrene immortalized and further transformed with SV40T, respectively. Four antibodies of two different classes were tested: 225 immunoglobulin G (IgG), 108.4 IgG, 96 immunoglobulin M (IgM), and 42 IgM. All four antibodies inhibited the anchorage-dependent and-independent, EGF-stimulated growth of 184A1N4 and 184A1N4-T cells, respectively, and this growth inhibition could be reversed by the addition of increasing concentrations of EGF. In contrast, the antibodies inhibited the anchorage-dependent and-independent growth of MDA-468 cells in the absence of exogenous EGF suggesting that the antibo...