Effects of aging and caloric restriction on IGF-I, IGF-I receptor, IGFBP-3 and IGFBP-4 gene expression in the rat stomach and colon

The purpose of this study was to determine the effects of aging and caloric restriction (CR) on insulin-like growth factor-I (IGF-I), IGF-I receptor (IGF-IR), IGF-binding protein-3 (IGFBP-3) and IGFBP-4 expression in the stomach and colon of male Fischer 344 rats. Stomach and colonic RNA were prepared from ad libitum (AL) fed or long-term CR rats. Stomach IGF-I, IGFBP-3 and IGFBP-4 mRNA levels increased significantly (P相似文献   

Insulin-like growth factor (IGF)-I, IGF-I receptor, and IGF binding protein-3 messenger ribonucleic acids and protein in corpora lutea from prostaglandin F(2alpha)-treated gilts

Insulin-like growth factor-I (IGF-I) is produced within the porcine corpus luteum (CL) and is thought to play an autocrine/paracrine role in CL development/function during the early luteal phase. This study examines the hypotheses that the luteolytic actions of prostaglandin F(2alpha) (PGF(2alpha)) during the early luteal phase may involve either a decrease in IGF-I or IGF receptor (IGF-IR), or an increase in IGF binding protein (IGFBP)-3, expression, any of which could interfere with the luteotropic actions of IGF-I in this tissue. Cycling gilts were treated twice daily with PGF(2alpha) (or saline) on Days 5-9 of the cycle to induce premature luteolysis. CL were collected on Days 6-9, and RNA, protein, or progesterone was extracted. By slot blot analysis, steady-state levels of IGF-I and IGFBP-3 mRNA were not different in PGF(2alpha)-treated vs. control animals; however, IGF-IR mRNA was increased in treated animals on Day 9. No changes in IGF-I content (ng/CL measured by RIA) were observed with respect to treatment. According to ligand blot analysis, the levels of IGFBP-3 increased on Day 6 and decreased on Days 8-9, while IGFBP-2 was higher on Days 6-7 and decreased on Day 9 in treated animals. IGF-IR levels, determined from Western blots, were higher on Day 7 (P < 0.05) and lower on Day 9 in PGF(2alpha)-treated animals vs. control animals (P < 0.05). In conclusion, PGF(2alpha)-induced premature luteolysis was associated with an increase in steady-state levels of IGF-IR mRNA, but it did not appear to be linked to changes in mRNA levels for IGF-I or IGFBP-3. However, since IGFBP-2 and -3 protein levels increased early in the treatment period (Days 6-7), it is possible that they may mediate the luteolytic actions of PGF(2alpha) by sequestering IGF-I and preventing its interaction with the IGF-IR.     

Expression of insulin-like growth factor binding protein-4, insulin-like growth factor-I receptor, and insulin-like growth factor-I in the mouse uterus throughout the estrous cycle

Recent evidence suggests that a regulated insulin-like growth factor (IGF) system mediates the effects of estrogen, promoting the proliferation and differentiation of specific uterine cell types throughout the estrous cycle and during gestation in the rodent. Previous studies have shown that IGFs are differentially expressed in the mouse uterus during the periimplantation period. In the current study, we examined the expression of IGF binding protein-4 (IGFBP-4), IGF-I receptor (IGF-IR), and IGF-I in the mouse uterus throughout the estrous cycle. Ligand blot analysis was conducted on uterine homogenates using [125I]IGF-I. IGFBP-4 was detected in all uterine homogenates, varying in intensity throughout the estrous cycle. In situ hybridization studies at metestrus and diestrus demonstrated an intense IGFBP-4 mRNA signal in antimesometrial stromal cells between the luminal epithelium and the myometrium, but at proestrus and estrus, no IGFBP-4 signal was detected. No IGF-I mRNA was detected at any stage of the estrous cycle by in situ hybridization. However, by RT-PCR analysis, IGF-I mRNA was detected at all stages of the estrous cycle. RT-PCR analysis also showed IGF-IR mRNA throughout the estrous cycle. Using immunohistochemistry, IGF-IR immunostaining was detected throughout the estrous cycle and on days 2-7 of gestation, but was restricted to the glandular epithelium. These results suggest that uterine IGFBP-4 expression may not be dependent on uterine IGF-I expression. They also suggest that IGFBP-4 may play a role in uterine physiology independent of the inhibition of IGF-I action, and that IGF-IR is constitutively expressed in the mouse uterus.     

A possible role for insulin-like growth factor-binding protein-3 autocrine/paracrine loops in controlling hepatocellular carcinoma cell proliferation.

Hepatocellular carcinoma (HCC) is a common malignancy, but treatment outcomes have generally remained poor. Specific factors important for the pathogenesis of HCC are incompletely understood. Insulin-like growth factors (IGFs) are potent autocrine and paracrine mitogens for liver cancer cell proliferation, and their bioactivity is reduced by IGF-binding protein 3 (IGFBP-3). In the present study, we report that IGFBP-3 protein levels were either undetectable (28.5%) or low (71.5%) in human HCC samples examined compared with matched non-neoplastic liver tissue by Western blotting. IGFBP-3 was localized to nontumor liver cells by immunohistochemistry with greater immunointensity than neoplastic liver cells. Levels of type I receptor (IGF-IR) were found to be low in approximately 39% of human HCC samples examined compared with matched nontumor tissues. IGF-II was overexpressed in 32%, whereas IGF-I expression was decreased in 100% of HCC samples. In vitro studies revealed that IGF-I and IGF-II induced HepG2 cell proliferation in a dose-dependent manner. Treatment of HepG2 cells with either human recombinant IGFBP-3 (hrIGFBP-3) or IGF-II antibody led to a significant reduction in cell proliferation. Cotreating these cells with hrIGFBP-3 significantly attenuated the mitogenic activity of IGF-I. IGF-I-induced phosphorylation of IGF-IR beta subunit, IRS-1, mitogen-activated protein kinase, Elk-1, and Akt-1 as well as phosphatidylinositol 3'-kinase activity was significantly attenuated when hepG2 cells were pretreated with hrIGFBP-3. Our data indicate that loss of autocrine/paracrine IGFBP-3 loops may lead to HCC tumor growth and suggest that modulating production of the IGFs, IGFBP-3, and IGF-IR may represent a novel approach in the treatment of HCC.     

Alteration of the insulin-like growth factor axis during in vitro differentiation of the human osteosarcoma cell line HOS 58

The insulin-like growth factors I and II (IGF-I, IGF-II), their receptors, and high affinity binding proteins (IGFBPs) represent a family of cellular modulators that play essential roles in the development and differentiation of cells and tissues including the skeleton. Recently, the human osteosarcoma cell line HOS 58 cells were used as an in vitro model of osteoblast differentiation characterized by (i) a rapid proliferation rate in low-density cells that decreased continuously with time of culture and (ii) an increasing secretion of matrix proteins during their in vitro differentiation. In the present paper, HOS 58 cells with low cell density at early time points of the in vitro differentiation (i) displayed a low expression of IGF-I and -II; (ii) synthesized low levels of IGFBP-2, -3, -4, and -5, but (iii) showed high expression levels of both the type I and II IGF receptors. During the in vitro differentiation of HOS 58 cells, IGF-I and -II expressions increased continuously in parallel with an upregulation of IGFBP-2, -3, -4, and -5 whereas the IGF-I receptor and IGF-II/M6P receptor mRNA were downregulated. In conclusion, the high proliferative activity in low cell density HOS 58 cells was associated with high mRNA levels of the IGF-IR, but low concentrations of IGFBP-2. The rate of proliferation of HOS 58 cells continuously decreased during cultivation in parallel with a decline in IGF-IR expression, but increase of mitoinhibitory IGFBP-2. These data are indicative for a role of the IGF axis during the in vitro differentiation of HOS 58 cells.     

Involvement of ligand occupancy in Insulin-like growth factor-I (IGF-I) induced cell growth in osteoblast like MC3T3-E1 cells

Growth factors and matrix proteins regulate the proliferation and differentiation of osteoblasts. The insulin-like growth factor (IGF) system comprises IGF-I, IGF-II, and six high-affinity IGF-binding proteins (IGFBPs). IGFs stimulate cell growth in many types of tissue; IGF-binding proteins regulate cellular actions and can affect cell growth. IGF-I is involved in differentiation, proliferation, and matrix formation in osteoblasts; IGFBP-5 is associated with the extracellular matrix (ECM) and can potentiate the actions of IGF-I. We investigated the effect of ECM proteins on the responses of MC3T3-E1 osteoblast cells to IGF-I and IGFBP-5. In addition, because extracellular signal-regulated kinases 1 and 2 (Erk 1/2) affect cell growth, we evaluated the effects of IGFBP-5 on Erk 1/2 phosphorylation in MC3T3-E1 cells. IGF-I caused an increase in IGFBP-5 expression in cultured MC3T3-E1 cells, and IGF-I plus IGFBP-5 significantly increased cell growth. Likewise, the addition of IGF-I and IGFBP-5 to cultured MC3T3-E1 cells increased the synthesis of the ECM proteins osteopontin (OPN) and thrombospondin-1 (TSP-1), which can bind to alphaVbeta3 integrin receptors on the cell surface. By contrast, the addition of an antibody against ECM proteins inhibited the effects of OPN and TSP-1 on IGFBP-5 expression. The stimulatory effect of IGFBP-5 was mediated via Erk 1/2 activation. These data suggest that IGFBP-5 regulates Erk 1/2 phosphorylation in cultured MC3T3-E1 cells via ECM proteins that may ultimately stimulate the growth of osteoblasts. We determined whether occupation of the alphaVbeta3 integrin receptor affects IGF-I receptor (IGF-IR)-mediated signaling and function in MC3T3-E1 osteoblast cells. Occupation of the alphaVbeta3 integrin receptor with ECM proteins induced IGF-I-stimulated IGF-IR phosphorylation. Conversely, in the presence of the alphaVbeta3-specific disintegrin echistatin, IGF-I-stimulated IGF-IR activation was inhibited. IGF-I-stimulated IGF-IR phosphorylation was accompanied by IRS-1 phosphorylation and MAPK activation. However, these effects were attenuated by echistatin. Thus, occupancy of the alphaVbeta3 disintegrin receptor modulates IGF-I-induced IGF-IR activation and IGF-IR-mediated function in MC 3T3-E1 osteoblasts.     

Expression of Insulin-like Growth Factor Binding Protein-3 and Regulation of the Insulin-like Growth Factor-I Axis in Pig Testis

The stem cell niche is a complex unit comprising key components, such as the extracellular matrix and various paracrine factors, which regulate the differentiation of adult stem cells. In our previous study, we established pig spermatogonial stem cells (pSSCs) in culture and identified the expression of insulin-like growth factor binding protein-3 (IGFBP-3) in pSSCs. The present study investigated not only the expression of IGFBP-3, but also its possible role in pSSCs. In this study, IGFBP-3-expressing cells responded positively to protein gene product 9.5 (PGP9.5), which is a marker for pig spermatogonia. IGFBP-3 expression was significantly increased in 60-dayold pig testes. Additionally, the expression levels of insulinlike growth factor I (IGF-I) and its receptor (IGF-IR) were observed in pSSCs and pig Sertoli cells (pSCs). Furthermore, IGF-I treatment enhanced the proliferation of pSCs and pSSCs when they were co-cultured. Blocking the IGF-I pathway using a specific IGF-IR inhibitor dramatically reduced the proliferation of pSCs. In addition, when heparan sulfate was used to sequester IGFBP-3 from IGF-I binding, a significant increase in the proliferation of pSCs was observed. Exogenous IGF-I treatment also increased the expression level of IGFBP-3 in cultured pSSCs. Furthermore, pSSCs grew well in IGF-I-treated pSC conditioned media. In summary, IGF-I and IGF-IR signaling are important for the proliferation of pSCs, and the germ cell-derived IGFBP-3 had an inhibitory effect on the mitotic activity of IGF-I in pSCs.     

Insulin-like growth factor family and combined antisense approach in therapy of lung carcinoma

BACKGROUND: Perturbation in a level of any peptide from insulin-like growth factor (IGF) family (ligands, receptors, and binding proteins) seems to be implicated in lung cancer formation; IGF ligands and IGF-I receptor through their mitogenic and anti-apoptotic action, and the mannose 6-phosphate/insulin-like growth factor II receptor (M6-P/IGF-IIR) possibly as a tumor suppressor. MATERIALS AND METHODS: To determine the identity, role, and mutual relationship of IGFs in lung cancer growth and maintenance, we examined IGF's gene (by RT-PCR) and protein (by immunohistochemistry) expression in 69 human lung carcinoma tissues. We also examined IGF-I receptor numbers (Scatchard analysis) and IGF-II production and release (by Western blot) in IGF-II/IGF-IR mRNA positive and negative lung carcinomas. Finally, the potential role of IGF-IR and IGF-II as growth promoting factors in lung cancer was studied using antisense oligodeoxynucleotides that specifically inhibit IGF-IR and IGF-II mRNA. RESULTS: Thirty-two tumors were positive for IGF-I, 39 for IGF-II, 48 for IGF-IR, and 35 for IGFBP-4 mRNA. Seventeen tumors were concomitantly positive for all four IGFs, whereas 34 were positive for IGF-II, IGF-IR, and IGFBP-4 mRNA. An elevated amount of IGF-II peptide was secreted into the growth medium of cell cultures established from five different IGF-II/IGF-IR mRNA positive lung cancer tissues. The cells also expressed elevated numbers of IGF-IR. Nine IGF-II-negative and 19 IGF-II-positive lung cancers of different stages were selected, and M6-P/ IGF-II receptor was determined immunohistochemically. Most of the IGF-II-negative tumors were strongly positive for M6-P/IGF-IIR. IGF-II-positive tumors were mostly negative for M6-P/IGF-II receptors. Antisense oligodeoxynucleotides to IGF-II significantly inhibited, by 25-60%, the in vitro growth of all six lung cancer cell lines. However, the best results (growth inhibition of up to 80%) were achieved with concomitant antisense treatment (to IGF-IR and IGF-II). CONCLUSION: Our data suggest that lung cancer cells produce IGF-IR and IGF-II, which in turn stimulates their proliferation by autocrine mechanism. Cancer cell proliferation can be abrogated or alleviated by blocking the mRNA activity of these genes indicating that an antisense approach may represent an effective and practical cancer gene therapy strategy.     

Characterization and regulation of insulin-like growth factor binding proteins in human hepatic stellate cells

Cultured hepatic stellate cells (HSCs), the cell type primarily involved in the progression of liver fibrosis, secrete insulin-like growth factor-I (IGF-I) and IGF binding protein (IGFBP) activity. IGF-I exerts a mitogenic effect on HSCs, thus potentially contributing to the fibrogenic process in an autocrine fashion. However, IGF-I action is modulated by the presence of specific IGFBPs that may inhibit and/or enhance its biologic effects. Therefore, we examined IGFBP-1 through IGFBP-6 mRNA and protein expression in HSCs isolated from human liver and activated in culture. Regulation of IGFBPs in response to IGF-I and other polypeptide growth factors involved in the hepatic fibrogenic process was also assessed. RNase protection assays and ligand blot analysis demonstrated that HSCs express IGFBP-2 through IGFBP-6 mRNAs and release detectable levels of IGFBP-2 through IGFBP-5. Because IGF-I, platelet-derived growth factor-BB (PDGF-BB), and transforming growth factor-β (TGF-β) stimulate HSC proliferation and/or matrix production, we tested their effect on IGFBPs released by HSCs. IGF-I induced IGFBP-3 and IGFBP-5 proteins in a time-dependent manner without an increase in the corresponding mRNAs. IGFBP-4 protein levels decreased in response to IGF-I. TGF-β stimulated IGFBP-3 mRNA and protein but decreased IGFBP-5 mRNA and protein. In contrast, PDGF-BB failed to regulate IGFBPs compared with controls. Recombinant human IGFBP-3 (rhIGFBP-3) was then tested for its effect on IGF-I-induced mitogenesis in HSCs. rhIGFBP-3 inhibited IGF-I-stimulated DNA synthesis in a dose-dependent manner, with a peak effect observed at 25 nM IGFBP-3. Because TGF-β is highly expressed in cirrhotic liver tissue, we determined whether IGFBP-3 mRNA expression is increased in liver biopsies obtained from patients with an active fibroproliferative response due to viral-induced chronic active hepatitis. In the majority of these samples, IGFBP-3 mRNA was increased compared with normal controls. These findings indicate that human HSCs, in their activated phenotype, constitutively produce IGFBPs. IGF-I and TGF-β differentially regulate IGFBP-3, IGFBP-4, and IGFBP-5 expression, which, in turn, may modulate the in vitro and in vivo action of IGF-I. J. Cell. Physiol. 174:240–250, 1998. © 1998 Wiley-Liss, Inc.     

Insulin-like growth factor (IGF) binding protein-3 regulation of IGF-I is altered in an acidic extracellular environment.

While extracellular acidification within solid tumors is well-documented, how reduced pH impacts regulation of insulin-like growth factor-I (IGF-I) has not been studied extensively. Because IGF-I receptor binding is affected by IGF binding proteins (IGFBPs), we examined how pH impacted IGFBP-3 regulation of IGF-I. IGF-I binding in the absence of IGFBP-3 was diminished at reduced pH. Addition of IGFBP-3 reduced IGF-I cell binding at pH 7.4 but increased surface association at pH 5.8. This increase in IGF-I binding at pH 5.8 corresponded with an increase in IGFBP-3 cell association. This, however, was not due to an increase in affinity of IGFBP-3 for heparin at reduced pH although both heparinase III treatment and heparin addition reduced IGFBP-3 enhancement of IGF-I binding. An increase in IGF-I binding to IGFBP-3, though, was seen at reduced pH using a cell-free assay. We hypothesize that the enhanced binding of IGF-I at pH 5.8 is facilitated by increased association of IGFBP-3 at this pH and that the resulting cell associated IGF-I is IGFBP-3 and not IGF-IR bound. Increased internalization and nuclear association of IGF-I at pH 5.8 in the presence of IGFBP-3 was evident, yet cell proliferation was reduced by IGFBP-3 at both pH 5.8 and 7.4 indicating that IGFBP-3-cell associated IGF-I does not signal the cell to proliferate and that the resulting transfer of bound IGF-I from IGF-IR to IGFBP-3 results in diminished proliferation. Solution binding of IGF-I by IGFBP-3 is one means by which IGF-I-induced proliferation is inhibited. Our work suggests that an alternative pathway exists by which IGF-I and IGFBP-3 both associate with the cell surface and that this association inhibits IGF-I-induced proliferation.     

Insulin-like growth factor-binding protein-3 activates a phosphotyrosine phosphatase. Effects on the insulin-like growth factor signaling pathway

The proliferative action of insulin-like growth factors (IGF-I and -II) is mediated via the type I IGF receptor (IGF-IR) and is modulated by their association with high affinity binding proteins, IGFBP-1 to -6. We recently found that, in addition to its ability to bind IGFs, IGFBP-3 also inhibits IGF-IR activation independently of IGF binding and without interacting directly with IGF-IR. Here, we show that IGFBP-3 is capable of blocking the signal triggered by IGFs. Breast carcinoma-derived cells (MCF-7) were stimulated by des(1-3)IGF-I or [Gln(3),Ala(4),Tyr(15),Leu(16)]IGF-I, two IGF analogues with intact affinity for IGF-IR, but with weak or virtually no affinity for IGFBPs, then incubated with IGFBP-3. The activated IGF-IR was desensitized through reversal of its autophosphorylation, following which both phosphatidylinositol 3-kinase and p42(MAPK) activities were depressed. Direct measurement of phosphotyrosine phosphatase activity and reconstitution experiments using tyrosine-phosphorylated insulin receptor substrate-1 (IRS-1) indicated that IGFBP-3 activated a phosphotyrosine phosphatase (PTPase). This action appeared to be peculiar to IGFBP-3 among the IGFBPs, since neither IGFBP-1 nor IGFBP-5 (structurally the closest to IGFBP-3), had any such effect. Several cell lines derived from normal or tumor cells responsive to IGF-I were used to show that IGFBP-3-stimulated PTPase is cell type-specific. Although the precise nature of the phosphatase remains to be determined, the results of this study demonstrate that IGFBP-3 stimulates a phosphotyrosine phosphatase activity that down-regulates the IGF-I signaling pathway, suggesting a major role for IGFBP-3 in regulating cell proliferation.     

An in situ hybridization study of the insulin-like growth factor system in developing condylar cartilage of the fetal mouse mandible

The objective of this study was to investigate the involvement of the insulin-like growth factor (IGF) system in the developing mandibular condylar cartilage and temporomandibular joint (TMJ). Fetal mice at embryonic day (E) 13.0-18.5 were used for in situ hybridization studies using [35S]-labeled RNA probes for IGF-I, IGF-II, IGF-I receptor (-IR), and IGF binding proteins (-BPs). At E13.0, IGF-I and IGF-II mRNA were expressed in the mesenchyme around the mandibular bone, but IGF-IR mRNA was not expressed within the bone. At E14.0, IGF-I and IGF-II mRNA were expressed in the outer layer of the condylar anlage, and IGF-IR mRNA was first detected within the condylar anlage, suggesting that the presence of IGF-IR mRNA in an IGF-rich environment triggers the initial formation of the condylar cartilage. IGFBP-4 mRNA was expressed in the anlagen of the articular disc and lower joint cavity from E15.0 to 18.5. When the upper joint cavity was formed at E18.5, IGFBP-4 mRNA expression was reduced in the fibrous mesenchymal tissue facing the upper joint cavity. Enhanced IGFBP-2 mRNA expression was first recognized in the anlagen of both the articular disc and lower joint cavity at E16.0 and continued expression in these tissues as well as in the fibrous mesenchymal tissue facing the upper joint cavity was observed at E18.5. IGFBP-5 mRNA was continuously expressed in the outer layer of the perichondrium/fibrous cell layer in the developing mandibular condyle. These findings suggest that the IGF system is involved in the formation of the condylar cartilage as well as in the TMJ.     

Expression of Insulin-Like Growth Factor System Genes in Liver Tissue During Embryonic and Early Post-Hatch Development in Duck (Anas platyrhynchos Domestica)

The IGF system is one of the most important endocrine and paracrine growth factor systems that regulate fetal and placental growth, whereas the liver is the principal source of circulation IGF-I. In the present study, expression of IGF-I, IGF type-I receptor (IGF-IR), and IGF binding protein (IGFBP)-3 genes was quantified by RT-PCR in the liver tissue on days 13, 17, 21, 25, and 27 of embryonic development, as well as at 7 days post-hatching (PH) in meat-type Gaoyou ducks and egg-type Jinding ducks. The results showed that IGF-I mRNA could be detected as early as on E 13d, but the expression level was low throughout embryonic development before increasing dramatically by E 27d and 7 days PH in both duck breeds. However, Gaoyou ducks exhibited higher IGF-I mRNA level than Jinding ducks, and the differences were significant on E 13d, E 21d, and at 7 days PH. Expression of IGF-IR in liver increased gradually in the former stages of the embryonic development, reaching its highest point on E 21d, and then declined up until 7 days PH. The expression pattern of IGFBP-3 gene was similar to that of IGF-IR gene, increasing significantly from E 17d. The expression peak appeared on E 25d, then declined significantly just prior to hatching (day 27) and was followed by an increase at 7 days PH. In general, the expression level of IGF-IR and IGFBP-3 genes in Jinding ducks was higher than that in Gaoyou ducks. Inverse relationships were observed for the expression of IGF-I and IGF-IR, and IGF-I and IGFBP-3, whereas a positive relationship was observed for the expression of IGF-IR and IGFBP-3. Our data indicate a differential expression of selected genes that comprise the IGF system in the duck liver tissue during embryonic and early PH growth and development.     

Effect of insulin-like growth factor (IGF)-I and Des (1-3) IGF-I on the level of IGF binding protein-3 and IGF binding protein-3 mRNA in cultured porcine embryonic muscle cells

Insulin-like growth factor binding protein (IGFBP)-3 effects proliferation and differentiation of numerous cell types by binding to insulin-like growth factors (IGF) and attenuating their activity or by directly affecting cells in an IGF-independent manner. Consequently, IGFBPs produced by specific cells may affect their differentiation and proliferation. In this study we show that embryonic porcine myogenic cells, unlike murine muscle cell lines, produce significant quantities of a binding protein immunologically identified as IGFBP-3. Nonfusing cells subcultured from highly fused porcine myogenic cell cultures do not produce detectable IGFBP-3 protein or mRNA, thus suggesting the IGFBP-3 is produced by muscle cells in the porcine myogenic cell cultures. Treatment of porcine myogenic cultures with 20 ng of IGF-I or 20 ng of Des (1-3) IGF-I/ml serum-free media for 24 h results in a threefold reduction in the level of IGFBP-3 in conditioned media. This reduction is not affected by cell density over a sixfold range. Additionally, treatment for 24 h with 20 ng of IGF-I/ml media results in a sevenfold decrease in the steady-state level of IGFBP-3 mRNA. This IGF-I-induced decrease in IGFBP-3 mRNA level appears to be relatively unique to myogenic cells. IGF-I treatment also causes a fourfold increase in the steady-state level of myogenin mRNA. This increase in myogenin mRNA suggests that, as expected, IGF-I treatment accelerates differentiation of myogenic cells. The simultaneous decrease in IGFBP-3 mRNA and protein that accompanies IGF-I-induced myogenin expression suggests that differentiation of myogenic cells may be preceded or accompanied by decreased production of IGFBP-3.     

c-myb stimulates cell growth by regulation of insulin-like growth factor (IGF) and IGF-binding protein-3 in K562 leukemia cells

c-myb plays an important role in the regulation of cell growth and differentiation, and is highly expressed in immature hematopoietic cells. The human chronic myelogenous leukemia cell K562, highly expresses IGF-I, IGF-II, IGF-IR, and IGF-induced cellular proliferation is mediated by IGF-IR. To characterize the impact of c-myb on the IGF-IGFBP-3 axis in leukemia cells, we overexpressed c-myb using an adenovirus gene transfer system in K562 cells. The overexpression of c-myb induced cell proliferation, compared to control, and c-myb induced cell growth was inhibited by anti-IGF-IR antibodies. c-myb overexpression resulted in a significant increase in the expression of IGF-I, IGF-II, and IGF-IR, and a decrease in IGFBP-3 expression. By contrast, disruption of c-myb function by DN-myb overexpression resulted in significant reduction of IGF-I, IGF-II, IGF-IR, and elevation of IGFBP-3 expression. In addition, exogenous IGFBP-3 inhibited the proliferation of K562 cells, and c-myb induced cell growth was blocked by IGFBP-3 overexpression in a dose-dependent manner. The growth-promoting effects of c-myb were mediated through two major intracellular signaling pathways, Akt and Erk. Activation of Akt and Erk by c-myb was completely blocked by IGF-IR and IGFBP-3 antibodies. These findings suggest that c-myb stimulates cell growth, in part, by regulating expression of the components of IGF-IGFBP axis in K562 cells. In addition, disruption of c-myb function by DN-myb may provide a useful strategy for treatment of leukemia.     

Comparative effects of DHEA vs. testosterone, dihydrotestosterone, and estradiol on proliferation and gene expression in human LNCaP prostate cancer cells

Serum levels of the adrenal androgen dehydroepiandrosterone (DHEA) peak in men and women in the third decade of life and decrease progressively with age. Increasing numbers of middle-aged and older individuals consume over-the-counter preparations of DHEA, hoping it will retard aging by increasing muscle and bone mass and strength, decreasing fat, and improving immunologic and neurobehavioral functions. Because DHEA can serve as a precursor to more potent androgens and estrogens, like testosterone (T), dihydrotestosterone (DHT), and 17beta-estradiol (E2), supplemental DHEA use may pose a cancer risk in patients with nascent or occult prostate cancer. The steroid-responsive human LNCaP prostate cancer cells, containing a functional but mutated androgen receptor (AR), were used to compare effects of DHEA with those of T, DHT, and E2 on cell proliferation and protein and/or gene expression of AR, prostate-specific antigen (PSA), IGF-I, IGF-I receptor (IGF-IR), IGF-II, IGF-binding proteins-2, -3, and -5, (IGFBPs-2, -3, and -5), and estrogen receptor-beta (ERbeta). Cell proliferation assays revealed significant stimulation by all four steroids. DHEA- and E2-induced responses were similar but delayed and reduced compared with that of T and DHT. All four hormones increased gene and/or protein expression of PSA, IGF-IR, IGF-I, and IGFBP-2 and decreased that of AR, ERbeta, IGF-II, and IGFBP-3. There were no significant effects of hormone treatment on IGFBP-5 mRNA. DHEA and E2 responses were similar, and distinct from those of DHT and T, in time- and dose-dependent studies. Further studies of the mechanisms of DHEA effects on prostate cancer epithelial cells of varying AR status, as well as on prostate stromal cells, will be required to discern the implications of DHEA supplementation on prostatic health.     

Effects of a high plant protein diet on the somatotropic system and cholecystokinin in Atlantic salmon (Salmo salar L.)

To investigate the endocrine signalling from dietary plant protein on somatotropic system and gastrointestinal hormone cholecystokinin (CCK), two iso-amino acid diets based on either high plant or high fish meal protein were fed to Atlantic salmon. Salmon with an average starting weight of 641 ± 23 g (N = 180), were fed a fish meal (FM) based diet (containing 40% FM) or diets mainly consisting of blended plant proteins (PP) containing only 13% marine protein, of which only 5% was FM for 3 months. mRNA levels of target genes GH, GH-R, IGF-I, IGF-II, IGFBP-1, IGF-IR in addition to CCK-L, were studied in brain, hepatic tissue and fast muscle, and circulating levels of IGF-I in plasma of Atlantic salmon were measured. We detected reduced feed intake resulting in lower growth, weight gain and muscle protein accretion in salmon fed plant protein compared to a diet based on fish meal. There were no significant effects on the regulation of the target genes in brain or in hepatic tissues, but a trend of down-regulation of IGF-I was detected in fast muscle. Lower feed intake, and therefore lower intake of the indispensable amino acids, may have resulted in lower pituitary GH and lower IGF-I mRNA levels in muscle tissues. This, together with higher protein catabolism, may be the main cause of the reduced growth of salmon fed plant protein diet. There were no signalling effects detected either by the minor differences of the diets on mRNA levels of GH, GH-R, IGF-IR, IGF-II, IGFBP-1, CCK or plasma protein IGF-I.