Biomedical subjects

P Rotwein

Publications and source records attributed to P Rotwein.

At least 37 records · Page 2Linked to original sources

PubMed1996

Identification of the cAMP response element that controls transcriptional activation of the insulin-like growth factor-I gene by prostaglandin E2 in osteoblasts.

Insulin-like growth factor-I (IGF-I), a multifunctional growth factor, plays a key role in skeletal growth and can enhance bone cell replication and differentiation. We previously showed that prostaglandin E2 (PGE2) and other agents that increase cAMP activated IGF-I gene transcription in primary rat osteoblast cultures through promoter 1 (P1), the major IGF-I promoter, and found that transcriptional induction was mediated by protein kinase A. We now have identified a short segment of P1 that is essential for full hormonal regulation and have characterized inducible DNA-protein interactions involving this site. Transient transfections of IGF-I P1 reporter genes into primary rat osteoblasts showed that the 328-base pair untranslated region of exon 1 was required for a full 5.3-fold response to PGE2; mutation in a previously footprinted site, HS3D (base pairs +193 to +215), reduced induction by 65%. PGE2 stimulated nuclear protein binding to HS3D. Binding, as determined by gel mobility shift assay, was not seen in nuclear extracts from untreated osteoblast cultures, was detected within 2 h of PGE2 treatment, and was maximal by 4 h. This DNA-protein interaction was not observed in cytoplasmic extracts from PGE2-treated cultures, indicating nuclear localization of the protein kinase A-activated factor(s). Activation of this factor was not blocked by cycloheximide (Chx), and Chx did not impair stimulation of IGF-I gene expression by PGE2. In contrast, binding to a consensus cAMP response element (CRE; 5'-TGACGTCA-3') from the rat somatostatin gene was not modulated by PGE2 or Chx. Competition gel mobility shift analysis using mutated DNA probes identified 5'-CGCAATCG-3' as the minimal sequence needed for inducible binding. All modified IGF-I P1 promoterreporter genes with mutations within this CRE sequence also showed a diminished functional response to PGE2. These results identify the CRE within the 5'-untranslated region of IGF-I exon 1 that is required for hormonal activation of IGF-I gene transcription by cAMP in osteoblasts.

Animals↗

PubMed1996

Contrasting acute in vivo nuclear actions of growth hormone and prolactin.

Growth hormone (GH) and prolactin (PRL) exert long-term effects on cellular metabolism, growth, and development through changes in gene expression and protein biosynthesis that are initiated by hormone binding to specific cell-surface receptors. Recent studies have demonstrated that ligand-induced activation of both GH and PRL receptors leads to the tyrosine phosphorylation of multiple intracellular proteins by the identical non-receptor tyrosine kinase, JAK2. We have shown previously that in vivo administration of human recombinant GH rapidly stimulated the inducible transcription factors, Stats1, 3, and 5, and acutely altered gene transcription in the liver. Because human GH can bind to both lactogenic and somatogenic receptors with high affinity, in this study we have addressed the question of specificity of the hormonal response by examining the early nuclear events following a single injection of rat GH or rat PRL to hormone-deficient hypophysectomized female rats. We find that PRL stimulated tyrosine phosphorylation of Stat5, induced nuclear protein binding to the GH-responsive element of the serine protease inhibitor (Spi) 2.1 promoter, and activated Spi 2.1 gene expression. These acute actions of rat PRL were modest compared to the effects of rat GH. GH treatment induced tyrosine phosphorylation of several hepatic nuclear proteins, activated Stats1, 3, and 5, stimulated Spi 2.1 gene expression, and inhibited albumin gene transcription. All of the effects of rat GH paralleled responses to human GH that we have measured previously. Based on these results, it is likely that most of the actions of human GH in the liver are mediated by the GH receptor rather than by the PRL receptor. The diminished response to PRL may be secondary to the high density of short PRL receptor isoforms in the liver, which do not participate effectively in ligand-induced signal transmission.

Animals↗

PubMed1996

Insulin-like growth factor-II is an autocrine survival factor for differentiating myoblasts.

Recent studies indicate that insulin-like growth factor-II (IGF-II) acts as an autocrine differentiation factor for skeletal myoblasts in culture. IGF-II mRNA and protein are induced as early events in muscle differentiation, and the rate and extent of IGF-II secretion correlate with both biochemical and morphological differentiation. Here we show that IGF-II also functions as an essential survival factor during the transition from proliferating to differentiating myoblasts. Stably transfected C2 muscle cell lines were established in which a mouse IGF-II cDNA was expressed in the antisense orientation relative to the constitutively active Moloney sarcoma virus promoter. IGF-II antisense cells proliferated normally in growth medium containing 20% serum but underwent rapid death when placed in low serum differentiation medium. Death was accompanied by characteristic markers of apoptosis with more than 90% of cells showing DNA fragmentation within 12-16 h. Myoblast death was prevented by IGF-I, des [1-3] IGF-I, IGF-II, and insulin with a dose potency consistent with activation of the IGF-I receptor; death also could be blocked by the protein synthesis inhibitor, cycloheximide. Exogenous IGFs additionally stimulated passage through a single cell cycle and subsequently induced terminal differentiation. Cell survival and cell cycle progression also were enhanced by fibroblast growth factor-2 and platelet-derived growth factor-bb, but these peptides did not promote differentiation. Our results define a novel system for studying apoptotic cell death and its prevention by growth factors, underscore the importance of IGF action in minimizing inappropriate cell death, and indicate that shared signal transduction pathways may mediate myoblast survival in vitro.

Animals↗

PubMed1996

Promoter-dependent and -independent activation of insulin-like growth factor binding protein-5 gene expression by prostaglandin E2 in primary rat osteoblasts.

Insulin-like growth factor (IGF) action is mediated by high affinity cell surface IGF receptors and modulated by a family of secreted IGF binding proteins (IGFBPs). IGFBP-5, the most conserved of six IGFBPs characterized to date, uniquely potentiates the anabolic actions of IGF-I for skeletal cells. In osteoblasts, IGFBP-5 production is stimulated by prostaglandin E2 (PGE2), a local factor that mediates certain effects induced by parathyroid hormone, cytokines such as interleukin-1 and transforming growth factor-beta, and mechanical strain. In this study, we show that transcriptional and post-transcriptional events initiated by PGE2 collaborate to enhance IGFBP-5 gene expression in primary fetal rat osteoblast cultures. PGE2 treatment stimulated up to a 7-fold rise in steady-state levels of IGFBP-5 mRNA throughout 32 h of incubation. Analysis of nascent IGFBP-5 mRNA suggested that PGE2 had only a modest stimulatory effect on IGFBP-5 gene transcription, and transient transfection studies with IGFBP-5 promoter-reporter genes confirmed that PGE2 enhanced promoter activity by approximately 2-fold. Similar stimulatory effects were seen with forskolin. A DNA fragment with only 51 base pairs of the 5'-flanking sequence retained hormonal responsiveness, which may be mediated by a binding site for transcription factor AP-2 located at positions -44 to -36 in the proximal IGFBP-5 promoter. Incubation of osteoblasts with the mRNA transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole demonstrated that PGE2 enhanced IGFBP-5 mRNA stability by 2-fold, increasing the t1/2 from 9 to 18 h. The effects of PGE2 on steady-state IGFBP-5 transcripts were abrogated by preincubating cells with cycloheximide, indicating that the effects of PGE2 on both gene transcription and mRNA stability required ongoing protein synthesis. Therefore, both promoter-dependent and -independent pathways converge to enhance IGFBP-5 gene expression in response to PGE2 in osteoblasts.

Animals↗

PubMed1996

Overexpression of insulin-like growth factor-II induces accelerated myoblast differentiation.

Previous studies have shown that exogenous insulin-like growth factors (IGFs) can stimulate the terminal differentiation of skeletal myoblasts in culture and have established a correlation between the rate and the extent of IGF-II secretion by muscle cell lines and the rate of biochemical and morphological differentiation. To investigate the hypothesis that autocrine secretion of IGF-II plays a critical role in stimulating spontaneous myogenic differentiation in vitro, we have established C2 muscle cell lines that stably express a mouse IGF-II cDNA under control of the strong, constitutively active Moloney sarcoma virus promoter, enabling us to study directly the effects of IGF-II overproduction. Similar to observations with other muscle cell lines, IGF-II overexpressing myoblasts proliferated normally in growth medium containing 20% fetal serum, but they underwent enhanced differentiation compared with controls when incubated in low-serum differentiation medium. Accelerated differentiation of IGF-II overexpressing C2 cells was preceded by the rapid induction of myogenin mRNA and protein expression (within 1 h, compared with 24-48 h in controls) and was accompanied by an enhanced proportion of the retinoblastoma protein in an underphosphrylated and potentially active form, by a marked increase in activity of the muscle-specific enzyme, creatine phosphokinase, by extensive myotube formation by 48 h, and by elevated secretion of IGF binding protein-5 when compared with controls. These results confirm a role for IGF-II as an autocrine/paracrine differentiation factor for skeletal myoblasts, and they define a model cell system that will be useful in determining the biochemical mechanisms of IGF action in cellular differentiation.

Animals↗

PubMed1996

Insulin-like growth factor binding protein-5 modulates muscle differentiation through an insulin-like growth factor-dependent mechanism.

The insulin-like growth factor binding proteins (IGFBPs) are a family of six secreted proteins which bind to and modulate the actions of insulin-like growth factors-I and -II (IGF-I and -II). IGFBP-5 is more conserved than other IGFBPs characterized to date, and is expressed in adult rodent muscle and in the developing myotome. We have shown previously that C2 myoblasts secrete IGFBP-5 as their sole IGFBP. Here we use these cells to study the function of IGFBP-5 during myogenesis, a process stimulated by IGFs. We stably transfected C2 cells with IGFBP-5 cDNAs under control of a constitutively active promoter. Compared with vector-transfected control cells, C2 myoblasts expressing the IGFBP-5 transgene in the sense orientation exhibit increased IGFBP-5 levels in the extracellular matrix during proliferation, and subsequently fail to differentiate normally, as assessed by both morphological and biochemical criteria. Compared to controls, IGFBP-5 sense myoblasts show enhanced survival in low serum medium, remaining viable for at least four weeks in culture. By contrast, myoblasts expressing the IGFBP-5 antisense transcript differentiate prematurely and more extensively than control cells. The inhibition of myogenic differentiation by high level expression of IGFBP-5 could be overcome by exogenous IGFs, with des (1-3) IGF-I, an analogue with decreased affinity for IGFBP-5 but normal affinity for the IGF-I receptor, showing the highest potency. These results are consistent with a model in which IGFBP-5 blocks IGF-stimulated myogenesis, and indicate that sequestration of IGFs in the extracellular matrix could be a possible mechanism of action. Our observations also suggest that IGFBP-5 normally inhibits muscle differentiation, and imply a role for IGFBP-5 in regulating IGF action during myogenic development in vivo.

Animals↗

PubMed1996

Growth, differentiation, and survival: multiple physiological functions for insulin-like growth factors.

The insulin-like growth factors (IGFs), IGF-I and IGF-II, comprise a conserved pair of secreted proteins with diverse effects on growth, development, and metabolism. Insulin-like growth factor action is initiated upon binding to cell-surface receptors and is modulated through interactions with secreted IGF binding proteins (IGFBPs). The last decade has seen an explosion of new information about the physiological roles of the IGFs. In this review, we critically examine this information from biochemical, cell biological, and molecular genetic perspectives. We discuss the structures and functions of the two IGF receptors, outline the actions of the six IGFBPs, and summarize and interpret recent studies highlighting essential roles for components of the IGF system in the growth and development of the embryo and fetus, in tissue differentiation, in cell survival and proliferation, and in cancer. These results are discussed in the context of new opportunities for understanding the mechanisms of IGF action in multiple biological processes.

Animals↗

PubMed1996

Acute nuclear actions of growth hormone (GH): cycloheximide inhibits inducible activator protein-1 activity, but does not block GH-regulated signal transducer and activator of transcription activation or gene expression.

The mechanisms by which GH regulates gene expression to stimulate somatic growth and alter intermediary metabolism are unknown. We have shown previously that in vivo GH administration rapidly modifies the tyrosine phosphorylation of multiple hepatic nuclear proteins, including the inducible transcription factors, Stat1, Stat3, and (in this report) Stat5, and have found that hormone treatment also rapidly alters gene transcription in the liver. In this study, we have used the protein synthesis inhibitor, cycloheximide (CHX), to investigate which of the acute actions of GH are primary hormonal responses and which require concurrent protein synthesis. We found that many of the early changes in nuclear protein tyrosine phosphorylation and in nuclear protein-DNA binding after GH are not blunted by CHX. The activation of insulin-like growth factor I and Spi 2.1 gene expression and the inhibition of albumin transcription also are not blocked by CHX, suggesting that these effects are primary consequences of GH-activated signal transduction pathways. By contrast, CHX completely inhibits the induction of activator protein-1 DNA-binding activity by GH, indicating that this action is secondary to the stimulation of Fos and/or Jun protein biosynthesis. Our results support the idea that multiple primary and secondary signaling pathways contribute to the pleiotropic effects of GH on gene expression and provide a framework for delineating the mechanisms controlling the acute actions of GH.

Animals↗

PubMed1995

Structure and function of the mouse insulin-like growth factor binding protein 5 gene promoter.

The actions of insulin-like growth factors I and II (IGF-I and -II) are modulated by interactions with one or more of a family of secreted IGF binding proteins (IGFBPs). IGFBP-5, the most conserved of the six known IGFBPs, is a 252-amino-acid protein that has been shown both to potentiate and inhibit IGF action. In previous studies, we have cloned and characterized the mouse IGFBP-5 gene and demonstrated that it is expressed in a hierarchical pattern in different adult mouse tissues and during rodent embryonic development. In this report, we describe the initial analysis of the IGFBP-5 gene promoter. By transient gene transfer studies, we show the orientation-specific activity of DNA fragments containing from 31 to 4,100 bp from the 5'-flanking region of the mouse IGFBP-5 gene in directing expression of the heterologous reporter gene luciferase in Hep G2 cells. DNA fragments with only 156 bp of 5'-flanking sequence mediated over 60% of maximal promoter activity, and a segment containing the TATA box and the first 120 bp of exon 1 still conferred some promoter function. Within the highly active 156-bp region, we identified a 37-bp segment from -70 to -34 that exhibited specific binding in DNase I footprinting and gel-mobility shift experiments with Hep G2 nuclear protein extracts. The footprinted region, which is almost completely conserved in the rat and human IGFBP-5 genes, was responsible for at least 70% of the activity of the intact promoter, as evidenced by the deleterious consequences of small internal deletions within this sequence on promoter function.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Growth hormone rapidly activates rat serine protease inhibitor 2.1 gene transcription and induces a DNA-binding activity distinct from those of Stat1, -3, and -4.

Transcriptional regulation by growth hormone (GH) represents the culmination of signal transduction pathways that are initiated by the cell surface GH receptor and are targeted to the nucleus. Recent studies have demonstrated that the activated GH receptor can stimulate Stat1, a cytoplasmic transcription factor that becomes tyrosine phosphorylated and translocates to the nucleus, where it can interact with specific DNA sequences to modulate gene expression. GH also has been found to induce protein binding to a portion of the rat serine protease inhibitor (Spi) 2.1 gene promoter that is required for GH-induced transcription of Spi 2.1. Using GH-deficient hypophysectomized rats as a model, we show that GH treatment rapidly and potently induces both nuclear Spi 2.1 mRNA expression in the liver and specific nuclear protein binding to a 45-bp segment of the Spi 2.1 gene promoter. A GH-inducible gel-shifted complex appears within 15 min of systemic hormone administration and can be inhibited by an antiphosphotyrosine monoclonal antibody but is not blocked by a polyclonal antiserum to Stat1, Stat3, or Stat4, even though the nucleotide sequence contains two gamma interferon-activated sequence-like elements that could interact with STAT proteins. By Southwestern (DNA-protein) blot analysis, approximately 41- and 35-kDa GH-inducible proteins were detected in hepatic nuclear extracts with the Spi 2.1 DNA probe. Thus, a GH-activated signaling pathway stimulates Spi 2.1 gene expression through a unique mechanism that does not appear to involve known members of the STAT family of transcription factors.

Animals↗

PubMed1995

Rapid changes in gene expression after in vivo growth hormone treatment.

GH exerts long-lasting effects on somatic growth via changes in gene expression and protein biosynthesis that represent the culmination of signal transduction pathways initiated at the cell surface. Recent studies have demonstrated that ligand-induced activation of the GH receptor leads to the phosphorylation of multiple intracellular proteins, including latent cytoplasmic transcription factors, Stats 1 and 3. GH treatment also has been found to induce the expression of several genes in both in vitro and in vivo systems, and we have shown that GH rapidly activates insulin-like growth factor I (IGF-I) gene transcription in hypophysectomized rats. In this study, using the GH-deficient, hypophysectomized rat as a model, we have examined the earliest changes in gene expression that follow a single systemic injection of GH. We find that GH induces nascent nuclear IGF-I transcripts within 15 min of hormone treatment, a time course that parallels the GH-regulated appearance of nuclear c-fos messenger RNA (mRNA). By contrast, nuclear transcripts for c-jun did not increase in abundance until after 30 min after hormone injection, and the peak rise in c-jun mRNA was severalfold less than for c-fos or IGF-I. GH treatment also led to the acute inhibition of IGF binding protein-1 (IGFBP-1) and albumin gene expression. Nuclear IGFBP-1 mRNA levels declined to 60% of baseline at 30 min and to 30% at 60 min, in agreement with previous studies showing a reduction in IGFBP-1 transcription after GH. Nascent nuclear albumin transcripts also decreased in abundance after GH treatment to levels that were less than 20% of basal values at 30 and 60 min. Our results show that GH can acutely activate and inhibit gene expression in the liver. It is likely that these diverse effects of GH are mediated by multiple signal transduction pathways.

Animals↗

PubMed1995

Characterization of deoxyribonucleic acid-protein interactions at a growth hormone-inducible nuclease hypersensitive site in the rat insulin-like growth factor-I gene.

Many of the growth-promoting effects of GH are mediated by insulin-like growth factor-I (IGF-I), a highly conserved, 70-residue basic peptide. Previous studies have demonstrated that GH rapidly stimulates IGF-I expression in vivo, and our laboratory has identified a GH-regulated alteration in chromatin configuration, manifested as a hormonally induced deoxyribonuclease-I (DNase-I)-hypersensitive site in the second IGF-I intron. In the current study, we have used in vivo DNase-I footprinting to map this hormonally responsive chromatin domain to an approximately 350-nucleotide region and have identified DNA-protein interactions within the hypersensitive site by in vitro gel mobility shift experiments and DNase-I footprinting studies. DNA-protein binding was localized to two adjacent segments of 32 and 48 nucleotides. In 1 of these regions, protein-DNA contacts were also detected in vivo on guanine residues by dimethylsulfate footprinting. DNA-binding activity was present in GH-deficient rats, but was not modified by hormone treatment. Our results define a rapid and reversible genomic alteration in response to GH in a GH-regulated gene and delineate a target within chromatin for GH action.

Animals↗

PubMed1995

Rapid activation of rat insulin-like growth factor-I gene transcription by growth hormone reveals no changes in deoxyribonucleic acid-protein interactions within the second promoter.

Insulin-like growth factor-I (IGF-I) is a highly conserved 70-residue circulating peptide that mediates many of the systemic growth-promoting effects of GH. This laboratory has found previously that GH rapidly stimulates hepatic IGF-I transcription in hypophysectomized (hypox) rats by activating promoter 1, the major rat IGF-I gene promoter. In this study, the hormonal regulation of IGF-I expression through promoter 2, a minor promoter in most tissues but active in the liver, was investigated. Through use of a sensitive RNase protection assay, GH was shown to rapidly induce the accumulation of correctly initiated transcripts directed by this promoter in hepatic nuclei. Using in vitro DNase-I footprinting, six DNA-protein interactions were identified within promoter 2 with hepatic nuclear extracts from juvenile male hypox rats given a single ip injection of GH or saline 60 min before death. These DNA-protein-binding complexes also were investigated for specificity and for regulation by GH by gel mobility shift assays. All DNA-protein interactions were detected in hepatic nuclear protein extracts from hypox rats and did not change within 15-120 min after GH treatment. These results thus identify and characterize a series of constitutive nuclear protein-binding sites within the second rat IGF-I promoter that may be involved in mediating its transcriptional activity.

Animals↗

PubMed1995

Regulation of insulin-like growth factor I transcription by cyclic adenosine 3',5'-monophosphate (cAMP) in fetal rat bone cells through an element within exon 1: protein kinase A-dependent control without a consensus AMP response element.

Insulin-like growth factor I (IGF-I) is a locally synthesized anabolic growth factor for bone. IGF-I synthesis by primary fetal rat osteoblasts (Ob) is stimulated by agents that increase the intracellular cAMP concentration, including prostaglandin E2 (PGE2). Previous studies with Ob cultures demonstrated that PGE2 enhanced IGF-I transcription through selective use of IGF-I promoter 1, with little effect on IGF-I messenger RNA half-life. Transient transfection of Ob cultures with an array of promoter 1-luciferase reporter fusion constructs has now allowed localization of a potential cis-acting promoter element(s) responsible for cAMP-stimulated gene expression to the 5'-untranslated region (5'-UTR) of IGF-I exon 1, within a segment lacking a consensus cAMP response element. Our evidence derives from three principal observations: 1) a transfection construct containing only 122 nucleotides (nt) of promoter 1 and 328 nt of the 5'-UTR retained full PGE2-stimulated reporter expression; 2) maximal PGE2-driven reporter expression required the presence of nt 196 to 328 of exon 1 when tested within the context of IGF-I promoter 1; 3) cotransfection of IGF-I promoter-luciferase-reporter constructs with a plasmid encoding the alpha-isoform of the catalytic subunit of murine cAMP-dependent protein kinase (PKA) produced results comparable to those seen with PGE2 treatment, whereas cotransfection with a plasmid encoding a mutant regulatory subunit of PKA that cannot bind cAMP blocked PGE2-induced reporter expression. Deoxyribonuclease I footprinting of the 5'-UTR of exon 1 demonstrated protected sequences at HS3A, HS3B, and HS3D, three of six DNA-protein binding sites previously characterized with rat liver nuclear extracts. Of these three regions, only the HS3D binding site is located within the functionally identified hormonally responsive segment of IGF-I exon 1. These results directly implicate PKA in the control of IGF-I gene transcription by PGE2 and identify a segment of IGF-I exon 1 as being essential for this hormonal regulation.

Animals↗

PubMed1995

Control of insulin-like growth factor-II/mannose 6-phosphate receptor gene transcription by proximal promoter elements.

The insulin-like growth factor-II/cation-independent mannose 6-phosphate receptor (IGF-II/MPR) is a multifunctional protein that binds IGF-II and ligands containing a mannose 6-phosphate recognition marker. Recent studies have shown that this receptor plays a critical role in mammalian development, and that its expression is controlled by both epigenetic and tissue-specific factors. Our laboratory has cloned the 93-kilobase mouse gene and characterized its 48 exons. In this report we describe the structure and function of the IGF-II/MPR gene promoter. To study promoter function, a series of chimeric plasmids linking different segments of IGF-II/MPR 5' flanking DNA to the reporter gene, firefly luciferase, were transiently transfected into HepG2 and C3H 10T1/2 cells. Promoter activity was orientation-specific and was maximal (550- to 4250-fold above promoterless control) with a plasmid containing 266 base pairs (bp) of IGF-II/MPR DNA. The fusion gene accurately directed transcription as measured by ribonuclease protection assay using RNA extracted from transfected cells. DNA-protein binding studies by in vitro DNase I footprinting revealed an extended 54-bp footprint within the proximal promoter that contained two E-boxes and potential binding sites for transcription factors Sp1, NGF-IA, and related proteins. Gel mobility shift experiments with double-stranded oligonucleotides containing this region gave rise to several specific DNA-protein complexes, and the addition of specific antibodies indicated that proteins antigenically related to Sp1 and c-Myc were components of one or more of these bands. Deletion of this 54-bp segment led to an 8-fold decline in promoter activity, and its transfer to a heterologous promoter stimulated gene expression by nearly 7-fold. Mutational analyses indicated that each E box contributed to more than half of the enhancer's activity. These results define a strong minimal IGF-II/MPR promoter of no more than 266 bp and identify a 54-bp enhancer within this promoter fragment. Our observations thus represent a first step toward characterizing the developmental, epigenetic, and tissue-specific factors that control IGF-II/MPR gene expression.

Amino Acid Sequence↗

PubMed1995

In vivo growth hormone treatment rapidly stimulates the tyrosine phosphorylation and activation of Stat3.

The mechanisms by which GH regulates gene expression to alter growth and metabolism are unknown. We have demonstrated previously that in vivo GH treatment rapidly stimulates the tyrosine phosphorylation of multiple nuclear proteins and have identified the inducible transcription factor Stat1 (formerly Stat91) as one of the major GH-activated nuclear phosphoproteins. We now show that Stat3, a new member of the STAT (signal transducer and activator of transcription) family of transcription factors, is also phosphorylated on tyrosine residues and rapidly appears in the nucleus in response to GH. Activated Stat3 interacts with the naturally occurring c-sis-inducible element of the c-fos gene after GH treatment, as demonstrated by gel mobility shift assay, and is a component of gel-shifted bands A and B when the high affinity sis-inducible element is used as a probe. Our results suggest that multiple STAT proteins may mediate some of the pleiotropic effects of GH on gene expression.

Animals↗

PubMed1995

Rapid activation of insulin-like growth factor binding protein-5 gene transcription during myoblast differentiation.

Insulin-like growth factor binding proteins (IGFBPs) comprise a family of secreted proteins that bind insulin-like growth factors-I and -II (IGF-I and -II) with high affinity and potentially modulate their biological effects. We have demonstrated previously that IGFBP-5, the most conserved of the six known IGFBPs, is expressed in muscle cells in the developing embryo and during the terminal differentiation of several myogenic cell lines. In this study we show that an IGF-I analog that binds minimally to IGFBPs potently enhances the differentiation of the stringently controlled inducible C2 myoblast (C2l) cell line and identify IGFBP-5 as the sole IGFBP secreted during C2l differentiation. We find that induction of IGFBP-5 mRNA and protein is coincident with the onset of myogenin gene expression and occurs secondary to the rapid activation of IGFBP-5 gene transcription. By transient gene transfer experiments we demonstrate that a 1004 base pair segment of the IGFBP-5 promoter is very active in directing expression of the reporter gene luciferase in C2l myoblasts. A promoter fragment containing only 156 nucleotides of 5'-flanking DNA retained more than 70% of maximal activity and mediated at least part of the differentiation-dependent rise in IGFBP-5 gene transcription. Within this active segment are several potential binding sites for muscle-enriched transcription factors. Our results show that induction of IGFBP-5 expression is an early event in the myogenic differentiation of the C2l cell line and suggest that one function of this IGFBP is to modulate IGF-induced differentiation. C2l cells are thus an excellent in vitro model for elucidating the developmental factors that control IGFBP-5 gene transcription and action in skeletal muscle.

Animals↗

PubMed1994

Loss of the imprinted IGF2/cation-independent mannose 6-phosphate receptor results in fetal overgrowth and perinatal lethality.

Murine embryos that inherit a nonfunctional insulin-like growth factor-II/cation-independent mannose 6-phosphate receptor (Igf2r) gene from their fathers are viable and develop normally into adults. However, the majority of mice inheriting the same mutated allele from their mothers die around birth, as a consequence of major cardiac abnormalities. These mice do not express IGF2R in their tissues, are 25-30% larger than their normal siblings, have elevated levels of circulating IGF2 and IGF-binding proteins, and exhibit a slight kink in their tails. These results show that Igf2r is paternally imprinted and reveal that the receptor is crucial for regulating normal fetal growth, circulating levels of IGF2, and heart development.

Alleles↗