Biomedical subjects

P Rotwein

Publications and source records attributed to P Rotwein.

At least 55 records · Page 3Linked to original sources

PubMed1994

Rapid changes in nuclear protein tyrosine phosphorylation after growth hormone treatment in vivo. Identification of phosphorylated mitogen-activated protein kinase and STAT91.

Growth hormone (GH) plays a central role in regulating growth and intermediary metabolism in vertebrates, although the mechanisms by which GH initiates these actions are largely unknown. The GH receptor, a member of the cytokine receptor superfamily, does not demonstrate homology with any known tyrosine kinases. However, addition of GH to cells in vitro has been shown to stimulate tyrosine phosphorylation of various intracellular proteins including mitogen-activated protein kinases (MAP kinases) and the newly described Janus kinase, JAK2. Subsequent steps in GH-mediated signal transduction have not been delineated. In the present study, we have examined early events in GH action in vivo. Hypophysectomized juvenile male rats were treated with GH for 15, 30, or 60 min. Rat liver whole cell and nuclear extracts were prepared and analyzed via SDS-polyacrylamide gel electrophoresis and Western blotting techniques. GH rapidly stimulated the tyrosine phosphorylation of at least 8 nuclear proteins of 205, 91, 83, 80, 65, 53, 44, and 42 kDa, and caused the dephosphorylation of a single approximately 149-kDa protein. Using specific antibodies, we have identified three of these nuclear phosphoproteins as 42- and 44-kDa MAP kinases, and as STAT91, a 91-kDa component of the interferon-stimulated gene factor-3 protein complex. One consequence of the activation of STAT91 in the nucleus is the appearance of GH-stimulated DNA binding activity, as assessed by gel-mobility shift assay using an oligonucleotide containing a c-sis-inducible element from the c-fos promoter. These results show that nuclear protein tyrosine phosphorylation is a prominent early event in GH action in vivo and demonstrate a link between GH-stimulated signal transduction and target gene expression.

Animals↗

PubMed1994

The mouse insulin-like growth factor II/cation-independent mannose 6-phosphate (IGF-II/MPR) receptor gene: molecular cloning and genomic organization.

The mammalian insulin-like growth factor II/cation-independent mannose 6-phosphate receptor (IGF-II/MPR) is a multifunctional protein that binds both IGF-II and ligands containing a mannose 6-phosphate recognition marker through distinct high-affinity sites. This receptor plays an integral part in lysosomal enzyme transport, has a potential role in growth factor maturation and clearance, and may mediate IGF-II-activated signal transduction through a G-protein-coupled mechanism. Recent studies have shown that production of IGF-II/MPR mRNA and protein begins in the mouse embryo soon after fertilization and have demonstrated that the receptor gene is on mouse chromosome 17 and is maternally imprinted. In this paper, we report the cloning and characterization of the mouse IGF-II/MPR gene. The gene is 93 kb long, is composed of 48 exons, and codes for a predicted protein of 2482 amino acids. The extracellular part of the receptor is encoded by exons 1-46, with each of 15 related repeating motifs being determined by parts of 3-5 exons. A single fibronectin type II-like element is found in exon 39. The transmembrane portion of the receptor also is encoded by exon 46, and the cytoplasmic region by exons 46-48. The positions of exon-intron splice junctions are conserved between several of the repeats in the IGF-II/MPR and the homologous extracellular region of the gene for the other known lysosomal sorting receptor, the cation-dependent mannose 6-phosphate receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Organization, expression, and chromosomal location of the mouse insulin-like growth factor binding protein 5 gene.

Insulin-like growth factor binding proteins (IGFBPs) constitute a family of at least six secreted proteins that bind insulin-like growth factors I and II (IGF-I and -II) and are capable of modifying IGF actions on target cells. We previously have purified an approximately 29-kDa IGFBP that is secreted by myoblasts during their terminal differentiation, have identified the protein as mouse IGFBP-5, and have cloned its cDNA (James et al., 1993). In this study, we have characterized the mouse IGFBP-5 gene, established its pattern of expression in the adult mouse, and defined its chromosomal location. The 17-kb gene was isolated on overlapping cosmid and lambda clones, and the four exons encoding the 5914-bp mRNA were sequenced. The 5' end of the gene was mapped by solution-hybridization ribonuclease protection assay to two discrete sites in exon 1 that were separated by 21 bp. The relative use of each transcription start site was found to vary among different mouse tissues. By interspecies backcross mapping using progeny derived from matings of [(C57BL/6J X Mus spretus)F1 x C57BL/6J] mice, the IGFBP-5 gene was localized to the proximal region of chromosome 1 in tight linkage with fibronectin 1 (0 recombinants in 168 mice analyzed). Since this part of chromosome 1 shares homology with human chromosome 2q, and since fibronectin has been mapped to 2q34-q36, it is likely that human IGFBP-5 will reside on 2q as well. Characterization of the mouse IGFBP-5 gene now provides a starting point for studying the roles and regulation of this protein in development.

Amino Acid Sequence↗

PubMed1994

Identification of two clusters of mouse insulin-like growth factor binding protein genes on chromosomes 1 and 11.

The genes for insulin-like growth factor binding proteins (IGFBPs) encode secreted proteins that bind insulin-like growth factors I and II with high affinity and modulate their biological activities. In this report we have used interspecific backcross mapping and gene cloning to define the chromosomal locations of 4 mouse Igfbp genes. Igfbp1 and 3 are found in the proximal part of chromosome 1. In the human genome these two loci map within 20 kb of one another on chromosome 7p14-p12, and the genes are organized in a tail-to-tail configuration. Mouse Igfbp2 and 5 colocalize to a proximal region of chromosome 1 that is syntenic with human chromosome 2q33-q36, and the two genes are 5 kb apart in a tail-to-tail orientation. These results suggest an evolutionary scheme in which a primordial IGFBP gene duplicated to form a cluster that was later replicated to create second linkage group.

Animals↗

PubMed1994

Rapid nuclear actions of growth hormone.

The mechanisms by which growth hormone (GH) initiates its effect on growth are largely unknown. In this report we examine the acute actions of GH with a focus on the intracellular signaling pathways leading from the cell-surface GH receptor into the nucleus, and culminating in the activation of specific target genes. We show that in vivo GH treatment leads to the rapid appearance of tyrosine-phosphorylated nuclear proteins and the equally rapid induction of c-fos and insulin-like growth factor I gene transcription. A model is proposed for a GH-activated intracellular signal transduction pathway.

Animals↗

PubMed1994

Distinct expression patterns of insulin-like growth factor binding proteins 2 and 5 during fetal and postnatal development.

Insulin-like growth factors (IGFs), when isolated from serum or tissue fluids, are usually found as part of a protein complex which also contains one of several IGF binding proteins (IGFBPs). Although some IGFBPs have been shown to alter interactions of IGFs with their receptors in vitro and can modify the responses of cultured cells to exogenous IGFs, the in vivo functions of IGFBPs remain unclear. This study examines expression of a recently described IGFBP gene, IGFBP-5, in the rat embryo and fetus and in selected adult tissues. Embryonic IGFBP-5 messenger RNA (mRNA) can be detected as early as embryonic day 10.5 and has an mRNA expression pattern distinct from the previously characterized pattern of IGFBP-2 mRNA expression. Major sites of IGFBP-5 expression during early postimplantation stages of development include the notochord, the floor plate, regions of the surface ectoderm, muscle precursor cells, and specific axial regions of neuroepithelium. Later in development IGFBP-5 mRNA is found in several regions of the central nervous system, including the proliferative zone of the external granule layer of the cerebellum and the mitral neurons of the olfactory bulb, as well as in muscle precursor populations of the developing limb, and in most cells of the anterior pituitary. In addition, only a subset of pituicytes in the adult posterior pituitary express IGFBP-5, which provides the first evidence that this cell population is biochemically heterogeneous. Taken together, these data suggest functions for IGFBP-5 during development of several organ systems.

Aging↗

PubMed1994

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

Insulin-like growth factor-I (IGF-I) is an important mediator of prenatal and postnatal growth, but little is known about the control of IGF-I gene expression. Previously, we demonstrated that GH rapidly stimulates hepatic IGF-I transcription in vivo in hypophysectomized (hypox) rats. In this study, we show that GH induces IGF-I gene transcription through the major promoter, promoter 1, and identify and characterize DNA-protein interactions throughout the promoter. In vitro deoxyribonuclease-I footprinting was used to analyze 1711 nucleotides of promoter 1 and the entire 328-nucleotide 5'-untranslated region of exon 1, using hepatic nuclear protein extracts from male juvenile hypox rats given a single ip injection of GH or saline 60 min before death. Fourteen DNA-protein binding sites were identified, with 6 located in the highly conserved 5'-untranslated region of exon 1. These latter sites were further characterized for specificity and regulation by GH, using gel mobility shift assays. Two of these DNA-protein interactions were also detected by in vivo dimethylsulfate footprinting. All DNA-protein binding was seen using hepatic nuclear protein extracts from hypox rats and did not change within 15, 30, 60, or 120 min after treatment with GH. Our results thus define a series of constitutive DNA-protein interactions within the major rat IGF-I gene promoter that may be involved in mediating GH-activated nuclear signals to initiate IGF-I transcription.

Amino Acid Sequence↗

PubMed1993

A highly conserved insulin-like growth factor-binding protein (IGFBP-5) is expressed during myoblast differentiation.

Insulin-like growth factor-binding proteins (IGFBPs) are a family of secreted proteins that bind insulin-like growth factors I and II (IGFs I and II) and are capable of modulating IGF actions on target cells. We have shown previously that C2 myoblasts secrete a single approximately 29-kDa IGFBP during their terminal differentiation (Tollefsen, S. E., Lajara, R., McCusker, R. H., Clemmons, D. R., and Rotwein, P. (1989) J. Biol. Chem. 264, 13810-13817). In this study, we have purified the protein from C2 cell-conditioned media by conventional and IGF-affinity chromatography, cloned its cDNA by PCR-based and traditional library screening, and identified it as mouse IGFBP-5. The resultant 5561 nucleotide cDNA encodes a 252-amino acid mature protein (predicted M(r) approximately 28,400) that is 97% identical to rat and human IGFBP-5. In differentiating C2 myoblasts and in F3 azamyoblasts the > 6-kilobase IGFBP-5 mRNA accumulates concomitantly with induction of myogen mRNA, an early marker of muscle differentiation. Ligand blot analysis shows that IGFBP-5 protein is secreted within 12 h of the onset of differentiation in these cells and that it is the only IGFBP produced in several fusing skeletal muscle cell lines. In vivo, IGFBP-5 transcripts are expressed in a variety of mouse tissues including striated muscle, but, unlike other IGFBPs, it is barely detectable in liver. IGFBP-5 is more conserved than other IGFBPs in mammals; its conserved structure and sequence also extends to non-mammalian vertebrates. Hybridization of a mouse BP5 coding region probe to RNA from several chicken and Xenopus tissues demonstrated similarly sized transcripts in these species. A partial Xenopus cDNA is identical in 38/45 deduced amino acids to the mammalian proteins. Identification of an IGF-binding protein that is produced during myoblast differentiation provides a model system in which to study the potential modulatory role of IGFBPs in development.

Amino Acid Sequence↗

PubMed1993

Multifactorial regulation of IGF-I gene expression.

Insulin-like growth factor-I (IGF-I) is a highly conserved 70-residue circulating peptide with diverse biological effects. In mammals IGF-I is an essential mediator of normal postnatal growth and its expression is influenced by hormonal, nutritional, tissue-specific, and developmental factors. Recent studies have demonstrated that the IGF-I gene is more complicated than might have been predicted from its simple protein sequence. In rats and in humans the single-copy six-exon gene is transcribed by adjacent promoters into nascent RNAs with different 5' leader sequences that undergo both alternative RNA splicing and differential polyadenylation to yield multiple mature transcripts. These observations suggest that trophic agents may modulate expression of IGF-I at any of several nodal points. In this report we review several of the mechanisms responsible for regulating production of IGF-I in the rat. During neonatal development IGF-I gene transcription is progressively activated, leading to a rise in both hepatic IGF-I mRNA and in serum IGF-I. The induction of IGF-I expression is limited to mRNAs directed by promoter 1, the more 5' of two rat IGF-I gene promoters, and precedes the ontogenic appearance of liver growth hormone (GH) receptors, indicating that mechanisms independent of GH activate IGF-I expression during early postnatal life. By contrast, in adult GH-deficient rats, a single intraperitoneal injection of GH causes a prompt rise in IGF-I gene transcription that is mediated equivalently by promoters 1 and 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1993

Prostaglandin E2 rapidly stimulates insulin-like growth factor-I gene expression in primary rat osteoblast cultures: evidence for transcriptional control.

Osteoblast-enriched (Ob) cultures isolated from fetal rat bone synthesize insulin-like growth factor-I (IGF-I), which functions as a locally acting growth and differentiation factor in the skeleton. Consistent with prior studies demonstrating that IGF-I production is enhanced in bone by agents that induce cAMP, prostaglandin E2 (PGE2) stimulates both cAMP synthesis and IGF-I mRNA in Ob cells. However, little is known about how cAMP regulates IGF-I expression in this or any other cell system. In rat tissues, multiple mechanisms influence levels of IGF-I mRNA, including transcription from two promoters, differential RNA splicing and stability, and alternative RNA polyadenylation. To determine how cAMP influences IGF-I gene expression in Ob cultures, we examined the responses of these cells to treatment with PGE2. PGE2 rapidly enhanced the accumulation of both large and small IGF-I transcripts, with increases in IGF-I mRNA detected within 2 h of treatment and persisting for 24 h. Analysis of precursor RNA by a highly specific and sensitive ribonuclease protection assay demonstrated a rise in nascent IGF-I mRNA within 30 min of exposure to PGE2, with a peak stimulation of 4-fold above control levels seen by 2 h and levels remaining elevated for up to 24 h. IGF-I transcripts in Ob cells were directed only by promoter 1, the more 5' of the two rat IGF-I gene promoters. As additionally assessed using the RNA polymerase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole, PGE2 treatment had little effect on IGF-I mRNA stability. In aggregate, these studies show that in fetal rat Ob cultures, PGE2 enhances IGF-I gene expression primarily through transcriptional mechanisms that are limited to a single IGF-I gene promoter. Ob cells, therefore, may be an excellent model for determining how cAMP regulates IGF-I gene transcription.

Animals↗

PubMed1993

Amino acid substitutions in the intracellular part of the growth hormone receptor in a patient with the Laron syndrome.

By complementary DNA cloning we have identified two amino acid substitutions in the intracellular region of the human GH receptor in a child with growth failure and clinical features of the Laron syndrome. At the second position of codon 422 a G to T transversion changes a cysteine residue to phenylalanine, whereas at the first nucleotide of codon 561 an alteration from C to A leads to the substitution of threonine for proline. Direct analysis of exon 10 of the GH receptor gene showed that both nucleotide substitutions reside on the same chromosome and were inherited from the patient's mother. Evaluation of DNA from 10 additional prospectively recruited children with growth failure and a clinical picture similar to the index case did not reveal any nucleotide alterations in codons 422, 560, or 561. These observations represent the first demonstration of variation within the intracytoplasmic part of the human GH receptor and indicate that mutations occurring at multiple locations within the receptor gene may lead to the same clinical phenotype.

Amino Acid Sequence↗

PubMed1993

Transcriptional activation of the insulin-like growth factor-II gene during myoblast differentiation.

Insulin-like growth factor-II (IGF-II) is a secreted 67-amino acid peptide that functions principally as a prenatal growth regulator in mammals. To date, the mechanisms involved in the stimulation of IGF-II expression in the embryo and its attenuation after birth are unknown. Recent studies have shown that IGF-II mRNA and protein are induced during the terminal stages of muscle development in vitro, and that IGF-II may act as an autocrine differentiation agent for skeletal myoblasts. We now have investigated the regulation of IGF-II gene expression in muscle cells. Steady state levels of IGF-II mRNA increased by more than 30-fold during terminal differentiation of the C2 mouse myoblast cell line. Transcription run-on experiments using isolated muscle cell nuclei and direct analysis of nuclear RNA each demonstrated a greater than 10-fold rise in nascent IGF-II mRNA during cellular differentiation, and ribonuclease protection experiments showed that more than 95% of IGF-II mRNAs initiated in noncoding exon 3, implying that transcriptional activation occurs principally through promoter 3, the most 3' of the three mouse IGF-II gene promoters. Analysis of chromatin structure around the IGF-II gene in C2 cells revealed four major and four minor DNase-I-hypersensitive sites, but did not provide insight into the mechanisms of gene activation, since all sites were present in proliferating and differentiating cells. Gene transfer experiments showed that promoter 3 was at least 50-fold more active than promoter 1 or 2 in C2 cells, but the functional assessment of nearly 26 kilobases of additional DNA within the IGF-II locus by an "enhancer trap" approach did not delineate any chromosomal regions capable of mediating differentiation-specific gene activation. Our results demonstrate that muscle cells encode mechanisms for activating IGF-II gene transcription and suggest that these cells may be excellent models for identifying the developmentally regulated factors that control IGF-II gene expression.

Animals↗

PubMed1992

Chromatin changes accompany the developmental activation of insulin-like growth factor I gene transcription.

Insulin-like growth factor I (IGF-I) is a potent regulator of postnatal growth in mammals, yet little is known about the developmental control of IGF-I synthesis. We have investigated the regulation of IGF-I expression in the rat in order to gain insight into the mechanisms of growth factor induction during early postnatal life. Steady-state levels of liver IGF-I mRNA increased by more than 15-fold during the period from fetal day 18 to postnatal day 7 and reached 50% of adult values by day 14. Transcription run-on experiments using isolated hepatic nuclei and direct analysis of nuclear RNA each demonstrated a comparable rise in nascent IGF-I mRNA over the same time period. Over 90% of transcripts were directed by promoter 1, the more 5' of the two IGF-I gene promoters. By contrast IGF-II gene transcription rates and mRNA levels fell during the first 3 weeks after birth, and albumin expression rose slightly. Analysis of chromatin structure around the IGF-I gene revealed 15 DNase I-hypersensitive sites in adult rat liver in the 120 kilobases (kb) comprising the 6 exon gene and its flanking regions (8 sites within 10 kb at the 5'-end including exons 1-3, 5 sites in the 50-kb third intron, and 2 sites in the 15-kb fifth intron). During development there was a progressive appearance of DNase I-hypersensitive sites that coincided with activation of IGF-I gene expression. One site that became fully hypersensitive by postnatal day 7 was mapped by in vivo DNAse I footprinting to the proximal 200 nucleotides of promoter 1. Since serum IGF-I values rose from 10 to 120 micrograms/liter during the initial postnatal week, these results indicate that transcriptional mechanisms are principally responsible for the stimulation of IGF-I synthesis that occurs shortly after birth. Because discrete changes in chromatin organization coincided with induction of IGF-I gene transcription, it is likely that a series of developmentally modulated transcription factors are involved the activation process.

Animals↗

PubMed1992

Functional analysis of the rat insulin-like growth factor I gene and identification of an IGF-I gene promoter.

Insulin-like growth factor I (IGF-I) mediates many of the systemic growth-promoting effects of growth hormone and also functions as a locally acting growth stimulator. In mammals, IGF-I gene expression is complicated, as the gene is transcribed and processed into multiple mRNAs (ranging in length from less than 1 to nearly 7.5 kb) that encode at least two protein precursors. As a step toward understanding the regulation of IGF-I, we report the complete organization of the rat IGF-I gene, including identification of the structural determinants for all IGF-I mRNA species, and an initial functional analysis of its promoters. The gene is composed of 6 exons distributed over nearly 80 kb of chromosomal DNA and is structurally heterogeneous. Several transcription start sites were identified within IGF-I exons 1 and 2, adjacent to presumptive promoters 1 and 2, respectively, and at least three polyadenylation sites were mapped to exon 6. To test promoter function, fusion genes were constructed linking fragments of IGF-I DNA to a reporter plasmid. Chimeric genes containing at least 395 bp of DNA from the 5'-flanking region of exon 1 enhanced luciferase activity after transfection into the IGF-I-producing SK-N-MC cell line, while fusion plasmids containing up to 1,300 bp of DNA from the 5'-flanking region of exon 2 were inactive. Relative levels of IGF-I mRNAs containing exons 1 or 2 varied among different rat tissues, although in response to acute or chronic growth hormone treatment both classes of transcripts were induced coordinately in rat liver. These observations represent the first thorough characterization of a mammalian IGF-I gene, and provide a starting point for defining the mechanisms by which growth hormone and other trophic factors regulate IGF-I gene expression.

Amino Acid Sequence↗

PubMed1992

Growth hormone rapidly activates insulin-like growth factor I gene transcription in vivo.

Many of the growth-promoting properties of GH are mediated by insulin-like growth factor I (IGF-I), a highly conserved circulating 70-amino acid peptide. Recent studies have shown that multiple mechanisms influence IGF-I gene expression, including transcription from two promoters, alternative RNA splicing, and variable polyadenylation. In order to determine how GH regulates IGF-I gene expression we have analyzed the response of hypophysectomized rats to a single ip injection of recombinant GH. A rise in hepatic IGF-I mRNA was detected within 2 h of GH treatment, with peak values of more than 15-fold above untreated animals by 4 h, and a decline by 16 h. A coordinate increase was seen in all IGF-I mRNA splicing and polyadenylation variants, indicating that neither alternative RNA processing nor differential poly A addition were altered by GH. Transcription run-on experiments using isolated hepatic nuclei and direct analysis of nuclear RNA demonstrated a rise in nascent IGF-I mRNA within 30 min of GH treatment, with peak levels reaching more than 10-fold above background by 2 h and declining by 6 h. As determined by RNase protection assays, transcripts directed by each promoter were coordinately and equivalently activated after GH. A single GH-responsive DNase I hypersensitive site was mapped in chromatin to the second IGF-I intron. This site exhibited rapid kinetics of induction which mirrored the pattern of transcriptional stimulation after GH treatment. These experiments show that GH enhances IGF-I expression in vivo by predominantly transcriptional mechanisms. The rapid kinetics of IGF-I gene activation and the temporally associated chromatin changes demonstrate a direct link between a GH-dependent signal transduction pathway and nuclear events.

Animals↗

PubMed1991

Structure of the chicken insulin-like growth factor I gene reveals conserved promoter elements.

Insulin-like growth factor I plays a major role in cellular growth by mediating many of the actions of growth hormone. In mammals, IGF-I gene expression is complicated, as the single-copy gene is transcribed and processed into multiple mRNAs that encode at least two peptide precursors. Recent cDNA cloning studies have suggested that in chickens the organization of the gene might be simpler, and that comparative mapping might define essential features of IGF-I that are common to vertebrates. To achieve this goal, we have cloned and characterized the chicken IGF-I gene. The chicken gene is more compact than its mammalian homologues. It is composed of 4 exons which are distributed over 50 kilobases (kb) of chromosomal DNA and are transcribed and processed into mRNAs of 1.9 and 2.6 kb. As in the rat and human genes, the mature 70-amino acid IGF-I molecule is encoded within exons 2 and 3, while exons 1 and 4 contain NH2- and COOH-terminal extension peptides, respectively, and untranslated sequences. Both ends of the chicken gene are structurally heterogeneous. IGF-I mRNAs were found to have two regions of polyadenylation which mapped to exon 4 and were separated by approximately 0.7 kb. At the 5' end, several transcription initiation sites were identified within a 74-nucleotide portion of exon 1 that was found to be highly conserved between chicken and mammalian IGF-I genes. To test promotor function, chimeric genes were constructed linking fragments of chicken IGF-I 5'-flanking DNA to a promoterless reporter plasmid. Hybrid genes containing at least 0.6 kb of IGF-I enhanced expression of luciferase after transfection into SK-N-MC cells, a human line that synthesizes IGF-I mRNA. These observations represent the first thorough characterization of an IGF-I gene and provide an impetus for discerning the mechanisms by which IGF-I expression is regulated during growth, development, and differentiation.

Amino Acid Sequence↗

PubMed1991

Differential regulation of mannose 6-phosphate receptors and their ligands during the myogenic development of C2 cells.

The mammalian insulin-like growth factor II/cation-independent mannose 6-phosphate receptor (IGF-II/CIMPR) mediates both targeting and endocytosis of mannose 6-phosphate-containing proteins and binds insulin-like growth factor II (IGF-II). The cation-dependent mannose 6-phosphate receptor (CDMPR) lacks an IGF-II-binding site and participates only in the intracellular trafficking of lysosomal enzymes. During terminal differentiation of the myogenic C2 cell line, there is an increase in cell surface expression of the IGF-II/CIMPR in parallel with a rise in secretion of IGF-II (Tollefsen, S.E., Sadow, J.L., and Rotwein, P. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 1543-1547). In this study we show that IGF-II/CIMPR mRNA increases by more than 10-fold during the initial 48 h of C2 muscle differentiation with kinetics similar to the rise in IGF-II mRNA. Comparable levels of both mRNAs are expressed in C2 myotubes and in primary cultures of fetal muscle. By contrast, no change is observed in CDMPR transcript abundance during differentiation, and only a small, transient increase is seen in the enzymatic activities and mRNA levels of several lysosomal enzymes. The differential regulation of the two mannose 6-phosphate receptors during muscle differentiation suggests that they may serve distinct functions in development.

Amino Acid Sequence↗

PubMed1991

Insulin-like growth factors and their receptors in muscle development.

Several proteins involved in IGF action are expressed in C2 cells and their abundance was found to vary as a function of development. IGF-I and II mRNA levels rose 10 and 25-fold, respectively, during differentiation, and were accompanied by an increase in growth factor secretion. The accumulation of IGF-II in conditioned culture medium was much greater than that of IGF-I. There was also an increase in the number of IGF-I receptors and IGF-II/CIMPR on the cell surface during differentiation. The sustained rise in IGF-II/CIMPR expression appeared to be a consequence of a similar increase in its mRNA abundance. The mechanisms responsible for the transient increment in IGF-I receptor number were not assessed, although it is likely that the decline in IGF-I receptor content after 72 hours in differentiation medium was a consequence of down-regulation by the IGF-II that accumulated in the medium (35). In contrast to the 13-fold rise in IGF-II/CIMPR mRNA levels, transcript levels for the CDMPR remained constant during C2 cell development, enzymatic activities of two lysosomal enzymes did not change, and only a small increment was detected at a single time point in the expression of several lysosomal enzyme mRNAs. In addition, during C2 muscle differentiation, a novel IGF binding protein was induced. These results demonstrate modulation of several components of IGF signaling pathways in differentiating myoblasts, and argue for a local role for IGFs in muscle development.

Animals↗