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D LeRoith

Publications and source records attributed to D LeRoith.

At least 19 recordsLinked to original sources
PubMed1995

The role of the tyrosine kinase domain of the insulin-like growth factor-I receptor in intracellular signaling, cellular proliferation, and tumorigenesis.

Insulin and insulin-like growth factor (IGF-I) receptors are heterotetrameric proteins consisting of two alpha-and two beta-subunits and members of the transmembrane tyrosine kinase receptors. Specific ligand binding to the receptor triggers a cascade of intracellular events, which begins with autophosphorylation of several tyrosine residues of the beta-subunit of the receptor. The triple cluster in the tyrosine kinase domain of the beta-subunit is the earliest and major autophosphorylation site. Previous studies have shown that substitutions of these three tyrosines by phenylalanines of both insulin and IGF-I receptors practically abolish any activation of cellular signaling pathways. We have studied the effect of double tyrosine mutations on IGF-I induced receptor autophosphorylation, activation of Shc and IRS-1 pathways, and cell proliferation and tumorigenicity. Substitution of tyrosines 1131/1135 blocks any detectable autophosphorylation, whereas substitution of tyrosines 1131/1136 or 1135/1136 only reduces autophosphorylation levels in some clones by approximately 50%. Nevertheless, all the cells expressing IGF-I receptors with double tyrosine substitutions demonstrated markedly reduced signaling through Shc and IRS-1 pathways. In addition, they were unable to respond to IGF-I-stimulated cell growth in culture, and tumor formation in nude mice was abrogated. These data suggest that the presence of tyrosine 1131 or 1135 essential for receptor autophosphorylation, whereas the presence of each of these tyrosines is necessary for a fully functional receptor.

3T3 Cells↗
PubMed1995

Insulin-like growth factor-I stimulates tyrosine phosphorylation of endogenous c-Crk.

Crk, a cellular homolog of v-crk, is an SH2 and SH3 domain-containing adaptor protein related to Grb2 and Nck, two proteins which have been shown to be involved in growth factor signal transduction. Crk proteins have recently been found to associate with two guanine nucleotide releasing proteins, mSos and C3G, and thus appear to lie on the Ras pathway. We investigated whether Crk is a target for the insulin-like growth factor I (IGF-I) receptor tyrosine kinase. We show that IGF-I stimulates tyrosine phosphorylation of Crk II via stimulation of endogenous IGF-I receptors in both 293 cells and NIH-3T3 cells. IGF-I stimulated tyrosine phosphorylation of Crk II in a dose- and time-dependent manner. In 293 cells, which express both IGF-I and insulin receptors, insulin also induced a dose-dependent tyrosine phosphorylation of Crk II, but with somewhat reduced sensitivity, compared to IGF-I. In NIH 3T3 cells, IGF-I also stimulated tyrosine phosphorylation of a 45- kDa protein which co-immunoprecipitated with Crk II. These findings indicate that Crk II is an endogenous substrate of the IGF-I receptor tyrosine kinase and provide the first demonstration that a mitogenic growth factor induces tyrosine phosphorylation of endogenous c-Crk.

3T3 Cells↗
PubMed1995

Mutation of a conserved amino acid residue (tryptophan 1173) in the tyrosine kinase domain of the IGF-I receptor abolishes autophosphorylation but does not eliminate biologic function.

The amino acid sequence of the tyrosine kinase domain of the insulin-like growth factor-I (IGF-I) receptor is 84% identical to the sequence of the analogous region of the insulin receptor. A naturally occurring mutation of the tryptophan residue at position 1200 of the insulin receptor to serine results in impaired beta subunit autophosphorylation of wheat germ agglutinin-purified receptors, severely impaired thymidine incorporation and moderately reduced glycogen synthesis; however, glucose uptake was unaffected. To study the importance of this residue in IGF-I receptor function, we mutated the analogous tryptophan residue at position 1173 of the IGF-I receptor to serine and overexpressed the mutant receptor in NIH-3T3 cells. In cell lines overexpressing this mutant IGF-I receptor, beta subunit autophosphorylation was severely reduced. Additionally, the overexpressed mutant receptors exhibited a dominant-negative effect on IGF-I-stimulated autophosphorylation of endogenous mouse IGF-I receptors. Phosphorylation of insulin receptor substrate (IRS)-1 in intact cells by the mutant IGF-I receptors was similar to the level of IRS-1 phosphorylation seen in the parental NIH-3T3 cells, but there was no obvious dominant-negative effect on IRS-1 phosphorylation. Wheat germ agglutinin-purified mutant receptors were as active in phosphorylating poly-(Glu,Tyr) 4:1 as wild-type IGF-I receptors, suggesting that, in intact cells, additional factors are necessary in order for the IGF-I receptor to phosphorylate IRS-1. Thymidine incorporation was severely reduced in one clone overexpressing the mutant IGF-I receptor and abolished in a second clone. Glucose uptake in both clones was reduced to about half of that seen in a cell line over-expressing wild-type IGF-I receptors. Thus, we propose that the tryptophan residue at position 1173 of the IGF-I receptor is important in the regulation of autophosphorylation in vivo. This study again confirms that high levels of autophosphorylation are not required for mediation of all of the biologic activities of the IGF-I receptor.

3T3 Cells↗
PubMed1995

Insulin-like growth factors and cancer.

The insulin-like growth factor (IGF) family of peptides, binding proteins, and receptors are important for normal human growth and development and are involved in the specialized functions of most physiologic systems. Most members of the IGF system are expressed by different cancer cells and may play an important role in the propagation of these malignancies. New therapies aimed at modulating various components of the IGF system could affect the progression and metastasis of cancer.

Animals↗
PubMed1995

Cytokines and growth factors modulate cell growth and insulin-like growth factor binding protein secretion by the human salivary cell line (HSG).

The human salivary cell line (HSG) was investigated for the effect of various growth factors and cytokines on cellular proliferation and on the production of insulin-like growth factor binding proteins (IGFBPs). IGF-I increased cell growth by approximately 25%, and induced the appearances of three distinct protein bands on ligand blot of the cell culture. Two bands with molecular weights of 43 and 45 Kda, respectively, proved to be IGFBP-3 using a specific antibody, and the third was a 24 Kda species (probably, IGFBP-4). Similar IGFBPs were released by the cells following stimulation by EGF and insulin as well as following incubation with the IGF-I receptor antibody alpha IR3. Retinoic acid had an inhibitory effect (50%) on IGF-I-induced cellular proliferation and an attenuative effect on the 24 Kda band when it was combined with IGF-I, and to a lesser effect EGF; however, it enhanced IGFBP-3 production when incubated with IGF-I. The IGF-I receptor antibody had an agonistic effect on IGFBPs production when applied alone or together with IGF-I. TNF-alpha and INF-gamma had minimal effects on cell growth when added alone but when applied in combination, a marked inhibition of cellular proliferation was noted. These cytokines caused increased accumulation of IGFBP-3, -4, and -5. Addition of IGF-I to these cytokines enhanced the expression of these bands. These data demonstrate that growth factors and cytokines which modulate HSG cell growth, induce specific IGFBPs which may play a role in their effects on cell growth.

Cell Division↗
PubMed1995

Dissociation of mitogenesis and transforming activity by C-terminal truncation of the insulin-like growth factor-I receptor.

We have investigated the mitogenic and transforming ability of an IGF-I receptor with a 108-amino-acid C-terminal truncation in R- cells, which are 3T3-like cells derived from mouse embryos in which the IGF-I receptor genes have been disrupted by targeted homologous recombination. R- cells stably transfected with expression plasmids encoding either a wild-type or a truncated human IGF-I receptor were capable of growing in serum-free medium supplemented solely with IGF-I. This response was observed over a wide range of receptor levels. R- cells overexpressing the wild-type IGF-I receptor also formed colonies in soft agar, and colony formation was augmented by coexpression of the SV40 large T antigen. However, all the examined clones of R- cells expressing the truncated IGF-I receptor exhibited a dramatically impaired ability to grow in soft agar, even in the presence of the T antigen. The inability to form colonies in soft agar was not due to a quantitative impairment of signal transduction, because: (1) SV40-transformed cells with a physiological level of the wild-type IGF-I receptor did not respond to IGF-I with cell proliferation, but grew in soft agar; (2) R- cells stably transfected with both a truncated receptor and T antigen, on the contrary, responded with mitogenesis to IGF-I but could not form colonies in soft agar; (3) some clones with the truncated receptor expressed levels of receptor roughly 100-fold the level of wild-type cells; and (4) several parameters of IGF-I receptor signal transduction were not impaired in cells stably transfected with a truncated receptor. Furthermore, overexpression of an activated ras in cells with the truncated IGF-IR did not restore their ability to proliferate under anchorage-independent conditions. We conclude that the 108 amino acids of the IGF-I receptor are not essential for a mitogenic response to IGF-I, but are required for transformation (as assessed by the ability to grow in soft agar), indicating that these two functions can be dissociated at an intramolecular level. Moreover, although ras (activated) certainly plays a role in transformation, the transforming activity of the IGF-IR also requires signaling elements that are ras-dependent.

3T3 Cells↗
PubMed1995

The role of growth hormone, insulin-like growth factors (IGFs), and IGF-binding proteins in experimental diabetic kidney disease.

Early renal changes in type I diabetes are characterized by an increase in renal size, glomerular volume, and kidney function, and later by development of mesangial proliferation, accumulation of glomerular extracellular matrix, and increased urinary albumin excretion (UAE). Growth hormone (GH) and insulin-like growth factors (IGFs) have a long and distinguished history in diabetes mellitus, with possible participation in the development of long-term complications. In experimental diabetes in dwarf rats with isolated GH and IGF-I deficiency, a slower and lesser renal and glomerular hypertrophy is observed as compared with diabetic control animals with intact pituitary. Furthermore, diabetic dwarf rats with a diabetes duration of 6 months display a smaller increase in UAE, indicating that GH and IGF-I may be involved in the development of diabetic kidney changes. In line with this, administration of octreotide to streptozotocin (STZ)-diabetic animals with normal pituitary inhibits initial renal growth without affecting blood glucose levels, and 6 months' administration of octreotide to diabetic rats reduces long-term renal/glomerular hypertrophy and UAE. In addition, the initial increase in renal size and function in experimental diabetes is preceded by an increase in renal IGF-I, IGF-binding proteins (IGFBPs), and IGF-II/mannose-6-phosphate receptor (IGF-II/Man-6-P receptor) concentration. Finally, specific changes occur in renal GH-binding protein (GHBP) mRNA, IGF-I receptor mRNA, and IGFBP mRNA expression in long-term diabetes. In conclusion, the knowledge we have today indicates that GH and IGFs, through a complex system consisting of GHBP, IGFs, IGF receptors, and IGFBPs, may be responsible for both early and late renal changes in experimental diabetes.

Animals↗
PubMed1995

Endogenous plasma growth hormone and the occurrence of pregnancies in patients undergoing in-vitro fertilization and embryo transfer with ovarian stimulation.

This study was undertaken to investigate a possible relationship between endogenous secretion of growth hormone (GH) during ovarian stimulation and treatment outcome in patients undergoing in-vitro fertilization (IVF) and embryo transfer. Plasma samples obtained from 19 women who had successfully completed all stages of IVF/embryo transfer were analysed retrospectively. Based on the increase in GH during treatment, 11 GH responders and eight GH non-responders were identified. Mean daily GH concentrations for the GH responders and GH non-responders were 3.5 +/- 1.8 and 1.8 +/- 0.8, 5.4 +/- 2.3 and 0.5 +/- 0.2, and 9.0 +/- 1.9 and 0.7 +/- 0.1 ng/ml (P < 0.05) for days 10, 11 and 12 respectively. Plasma insulin-like growth factor-I slightly increased with treatment in both groups. No significant difference between these groups was found in relation to treatment duration, number of human menopausal gonadotrophin ampoules used, oestradiol peak values, and number of oocytes retrieved or fertilization rate. Seven of the 11 GH responder women conceived in comparison with one pregnancy among eight GH non-responder patients (P < 0.05). In view of the absence of differences in the clinical and laboratory parameters, we suggest that the occurrence of pregnancies among GH responder patients might be related to a positive local effect of GH or its mediators on uterine receptivity at the time of nidation.

Adult↗
PubMed1995

Inhibition of cellular proliferation by the Wilms' tumor suppressor WT1 is associated with suppression of insulin-like growth factor I receptor gene expression.

We have investigated the regulation of the insulin-like growth factor I receptor (IGF-I-R) gene promoter by the Wilms' tumor suppressor WT1 in intact cells. The levels of endogenous IGF-I-R mRNA and the activity of IGF-I-R gene promoter fragments in luciferase reporter constructs were found to be significantly higher in G401 cells (a Wilms' tumor-derived cell line lacking detectable WT1 mRNA) than in 293 cells (a human embryonic kidney cell line which expresses significant levels of WT1 mRNA). To study whether WT1 could suppress the expression of the endogenous IGF-I-R gene, WT1-negative G401 cells were stably transfected with a WT1 expression vector. Expression of WT1 mRNA in G401 cells resulted in a significant decrease in the rate of cellular proliferation, which was associated with a reduction in the levels of IGF-I-R mRNA, promoter activity, and ligand binding and with a reduction in IGF-I-stimulated cellular proliferation, thymidine incorporation, and anchorage-independent growth. These data suggest that a major aspect of the action of the WT1 tumor suppressor is the repression of IGF-I-R gene expression.

Cell Division↗
PubMed1995

Regulation of insulin-like growth factor I transcription by prostaglandin E2 in osteoblast cells.

Insulin-like growth factor I (IGF-I) is a widely expressed abundant autocrine and paracrine factor that regulates the proliferation and differentiation of a variety of cell types. Prostaglandin E2 (PGE2) is a potent stimulator of IGF-I synthesis in bone. We examined the regulation of IGF-I synthesis by PGE2 in osteoblast-enriched (Ob) cells from fetal rat calvaria. PGE2 treatment of Ob cells at 1 microM for 2 h resulted in a 5-fold increase in heterogeneous nuclear RNA levels, as measured by a reverse transcriptase-polymerase chain reaction assay, suggesting an increase in IGF-I gene transcription. RNase protection analysis was used to map the transcriptional start sites in the IGF-I gene that are used in Ob cells. Consistent with other extrahepatic tissues, initiation of transcription occurs primarily at three sites within the 5'-regions of exon 1 of the IGF-I gene. PGE2 treatment did not alter start site usage. The regions upstream of these transcriptional start sites were analyzed by transiently transfecting Ob cells with putative rat IGF-I promoter sequences ligated to a luciferase reporter gene. Constructs containing 1.4 kilobases of the 5'-regions regions of exons 1 and 2 had significant promoter activity. PGE2 treatment of transfected Ob cells increased luciferase activity 5-fold when a 1.4-kilobase exon 1 promoter fragment was tested. This increase in luciferase activity was time and dose dependent. Smaller regions of the exon 1 promoter sequence gave higher basal activity and were less responsive to PGE2. We conclude that regions involved in IGF-I regulation by PGE2 are contained within the IGF-I promoter.

Animals↗
PubMed1995

Growth inhibition of MCF-7 breast cancer cells by stable expression of an insulin-like growth factor I receptor antisense ribonucleic acid.

Insulin-like growth factors (IGFs) play an important role in cellular proliferation, and IGF action appears to be involved in tumorigenesis. To determine the role of the IGF-I receptor in breast cancer cell growth, we stably transfected MCF-7 breast cancer cells with a construct encoding an antisense RNA complementary to the region surrounding the translation initiation site of the IGF-I receptor messenger RNA (mRNA). Control cells were transfected with vector alone. Clones expressing the antisense RNA exhibited a 30% reduction in endogenous IGF-I receptor mRNA levels and a significant reduction in receptor protein levels, as measured by both ligand binding assays and Western blot analysis. Antisense-expressing clones expressed approximately 30,000 receptors/cell compared with approximately 48,000-58,000 receptors/cell in control (neo) cells (P < 0.05). Although endogenous RNA:RNA hybrids were demonstrable in antisense-expressing cells, our results suggest that the major effect of the antisense may be the reduction in mRNA levels and not via an inhibition of translation. The reduction in receptor expression reduced both IGF-I- and serum-stimulated cellular proliferation. The maximum cell number reached at 96 h in the presence of IGF-I (100 ng/ml) was significantly reduced in antisense-expressing clones (22,000-30,000) compared with that in control (neo) cells (39,000-42,000). Furthermore, IGF-I-induced c-fos gene expression was reduced by 30% in the clones expressing the antisense RNA. These results strongly support a role for the IGF-I receptor in the proliferation of human breast cancer cells and suggest that strategies using this type of technology may prove useful in cancer therapy.

Breast Neoplasms↗
PubMed1995

Localization of growth hormone receptor/binding protein messenger ribonucleic acid (mRNA) during rat fetal development: relationship to insulin-like growth factor-I mRNA.

Although GH plays a key role in postnatal growth, prenatal growth is thought to be GH independent. However, recent data has shown GH receptor/binding protein (GHR/BP) to be present in rat fetal tissues as early as fetal stage E12. The aim of the present study was to investigate tissue-specific production of the GHR/BP messenger RNA (mRNA) and its relationship to locally transcribed insulin-like growth factor-I (IGF-I) mRNA in the fetus. We have used in situ hybridization to localize GHR/BP and IGF-I mRNAs in 16.5-, 18.5-, and 20.5-day-old rat fetuses. Furthermore, because the two parameters of the IGF-I gene differentially respond to GH stimulation, we have also investigated the presence and localization of promoter-specific IGF-I mRNAs. We found the distribution of IGF-I and GHR/BP mRNAs to be widespread but distinct during the fetal stages examined. High levels of IGF-I mRNA were found in connective tissues or their precursors, including the dermis, perichondrium, and gut. In contrast, GHR/BP mRNA exhibited three distinct patterns of distribution. First, GHR/BP mRNA was found at epithelial sites adjacent to sites of IGF-I transcription. Second, GHR/BP and IGF-I mRNAs were found to colocalize in some connective tissues, but GHR/BP mRNA levels in these sites were often lower than at other sites (i.e. epithelial) of GHR/BP gene transcription. Third, GHR/BP mRNA was also found in regions remote from IGF-I mRNA, including the nerve ganglia and inner olfactory bulb. Using promoter-specific IGF-I RNA probes, we detected only promoter 1 transcripts in all fetal tissues examined. The only exception occurred in specialized epithelial cells of the cochlea where we detected high levels of both promoter 1- and 2-derived IGF-I transcripts. We have thus demonstrated a distinct distribution of GHR/BP and IGF-I mRNAs in the developing rat fetus with coordinate expression at some sites. These findings suggest a role of GH or a GH-like peptide, acting both directly and indirectly via IGF-I, in fetal growth and development.

Animals↗
PubMed1995

Single tyrosine substitution in the insulin-like growth factor I receptor inhibits ligand-induced receptor autophosphorylation and internalization, but not mitogenesis.

The tyrosine kinase domains of the insulin and insulin-like growth factor I (IGF-I) receptors play an essential role in signal transduction. After ligand binding, these receptors undergo autophosphorylation, with a cluster of three tyrosines (residues 1131, 1135, and 1136 in the IGF-I receptor) being the first to be phosphorylated. Mutation of the ATP-binding site or substitution of this triple tyrosine cluster in the catalytic domain blocks essentially all of the functions of these receptors. Using stably transfected NIH-3T3 cell lines, we studied the effect of a mutation of tyrosine 1131 of the triple tyrosine cluster of the IGF-I receptor to phenylalanine. This mutation significantly reduced IGF-I-induced beta-subunit autophosphorylation, whereas phosphorylation of the endogenous substrate IRS-1 was unaffected. Despite the reduction in autophosphorylation and receptor internalization, IGF-I-induced thymidine incorporation and cellular proliferation were unaffected. Thus, the extent of receptor autophosphorylation and internalization does not appear to be a limiting factor for IGF-I-stimulated mitogenesis.

3T3 Cells↗
PubMed1995

Expression of insulin-like growth factor binding proteins in the rat kidney: effects of long-term diabetes.

Recent studies have shown that the renal synthesis of insulin-like growth factor binding proteins (IGFBPs) is altered in insulin-deficient diabetes mellitus, suggesting that these changes may be implicated in the alterations in renal function and morphology that accompany diabetes. To investigate the time course and the precise cellular distribution of changes in IGFBP expression, we used quantitative in situ hybridization to analyze renal IGF-I and IGFBP-1 to -5 messenger RNA (mRNA) localization and levels from 2 days to 6 months after the onset of streptozotocin-induced diabetes. There was an immediate sharp decline in IGF-I mRNA levels in the outer medulla that persisted for up to 3 months and a much smaller reduction in IGF-I mRNA levels in the medullary thick ascending limbs (MTALs). In nondiabetic animals, IGFBP-1 mRNA is most abundant in the MTALs. Immediately after the induction of diabetes, however, there was a greater than 2-fold increase in cortical IGFBP-1 mRNA and a 75% decrease in IGFBP-1 mRNA in MTALs. These changes persisted for up to 6 months in the diabetic animals. In contrast, IGFBP-5 mRNA levels were increased in the outer medulla and decreased in the cortex of diabetic kidneys. No significant changes in renal IG-FBP-2 mRNA levels or distribution were noted, and changes in IG-FBP-3 and -4 mRNA levels were subtle. In summary, streptozotocin-induced diabetes is associated with very prominent and complex alterations in renal IGF system gene expression, including robust increases in cortical IGFBP-1 and profound decreases in cortical IG-FBP-5 mRNA and medullary IGF-I mRNA levels. The divergent changes in IGFBP-1 and -5 mRNA levels in cortex vs. outer medulla indicate that regulation of IGFBP mRNA levels is quite complex.

Albuminuria↗
PubMed1995

Modulation of insulin-like growth factor I (IGF-I) receptors and membrane-associated IGF-binding proteins in endometrial cancer cells by estradiol.

Insulin-like growth factor I (IGF-I) receptors and membrane-associated IGF-binding proteins (IGFBPs) were examined in Ishikawa endometrial cancer cells. Our findings suggest that about 95% of [125I]IGF-I is bound to membrane-associated IGFBPs rather than to IGF-I receptors. Specifically, [125I]IGF-I binding to cell membranes could be completely displaced by cold IGF-I or IGF-II, but not by insulin, suggesting that binding was primarily due to IGFBPs. This was confirmed by using [125I]des-(1-3)IGF-I as the ligand. Des-(1-3) IGF-I binds with high affinity to IGF-I receptors, but with markedly lower affinity to IGFBPs. [125I]Des-(1-3)IGF-I bound to Ishikawa cells was displaced by IGF-I, IGF-II, and insulin. These results suggest that measuring IGF-I receptor levels using labeled IGF-I may be misleading. Accordingly, we evaluated the differential binding of [125I]IGF-I and [125I]des-(1-3)IGF-I to study the involvement of the IGF system in the stimulation of Ishikawa cell growth by estradiol. IGF-I stimulates Ishikawa cell proliferation, but at low concentrations, and this stimulation is largely dependent on the presence of estradiol. Estradiol caused a 2.5-fold increase in IGF-I receptor levels. Moreover, estradiol reduced soluble IGFBP levels, presumably increasing the availability of IGFs for their receptors. This elevation in IGF-I receptor levels and the decrease in IGFBP levels were accompanied by a 3.5-fold increase in IGF-I receptor messenger RNA and a 2.5-fold decrease in IGFBP messenger RNAs. These experiments suggest that estradiol sensitizes endometrial cancer cells to the effects of IGFs by simultaneously elevating receptor levels and decreasing (potentially inhibitory) IGFBP levels.

Binding, Competitive↗