Kinetics and regulation of site-specific endonucleolytic cleavage of human IGF-II mRNAs.

Human insulin-like growth factor II (IGF-II) mRNA can be cleaved at a specific site in its 4 kb long 3'-UTR. This yields a stable 3' cleavage product of 1.8 kb consisting of a 3'-UTR and a poly(A) tail and an unstable 5' cleavage product containing the IGF-II coding region. After cleavage, the 5' cleavage product is targeted to rapid degradation and consequently is no longer involved in IGF-II protein synthesis. Cleavage is therefore thought to provide an additional way to control IGF-II gene expression. In this paper the kinetics and the efficiency of cleavage of IGF-II mRNAs are examined. The cleavage efficiency of IGF-II mRNAs carrying four different leaders (L1-L4) is enhanced in the highly structured leaders L1 and L3. Additionally, under standard cell culture conditions cleavage is a slow process that only plays a limited role in destabilisation and translation of the IGF-II mRNAs. However, in human Hep3B cells and CaCo2 cells which express IGF-II endogenously, cleavage is upregulated 3-5-fold at high cell densities. Regulated endonucleolytic cleavage of IGF-II mRNAs is restricted to cells in which IGF-II expression is related to specific cell processes.

The chum salmon insulin-like growth factor II promoter requires Sp1 for its activation by C/EBPbeta.

The chum salmon insulin-like growth factor II (IGF-II) gene is highly expressed in liver tissue. In this study we demonstrate that two transcription factors, Sp1 and C/EBPbeta, are involved in the enhanced expression of the salmon IGF-II gene. The presence of the fish homolog for C/EBPbeta in salmon liver RNA was confirmed by Northern blotting. The sIGF-II promoter was activated up to 20-fold by co-transfection with C/EBPbeta. The functional importance of four out of the five putative C/EBPbeta binding sites was demonstrated with mutational analysis in transient transfection assays. The transcription factor Sp1 binds to two sites within the salmon IGF-II promoter. Interestingly, mutation of the Sp1 binding sites decreases not only the basal IGF-II promoter activity but also the C/EBPbeta-induced transactivation. These results demonstrate that liver-enriched C/EBPbeta and ubiquitously expressed Sp1 each activate the sIGF-II promoter and that Sp1 is required for full transactivation of the sIGF-II gene by C/EBPbeta. This suggests that C/EBPbeta and Sp1 act in synergy.

Stabilization of cyclin D1 mRNA via the phosphatidylinositol 3-kinase pathway in MCF-7 human breast cancer cells.

Treatment of quiescent MCF-7 human breast cancer cells with either the polypeptide growth factors insulin-like growth factor-I (IGF-I) or epidermal growth factor (EGF), the steroid hormone estradiol (E2) or the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) results in increased steady-state levels of cyclin D1 mRNA and protein. Unexpectedly, this elevation of cyclin D1 expression by all of these agents is inhibited by the specific phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002. Since transcriptional activation of the cyclin D1 promoter by EGF, E2 and TPA is independent of PI3-K activity, these findings suggest a post-transcriptional role for PI3-K in the regulation of cyclin D1 expression. Here we show that inhibition of PI3-K by LY294002 decreases the half-life of the 4.5 kb cyclin D1 mRNA species. In contrast, the stability of the 1.5 kb cyclin D1 mRNA is not affected by PI3-K inhibition. PI3-K-mediated stabilization of mRNA is not a general phenomenon, since other rapidly regulated and unstable mRNAs, such as those encoding c-fos, c-jun and c-myc, are not stabilized upon activation of the PI3-K signaling pathway.

Distinct RNA structural domains cooperate to maintain a specific cleavage site in the 3'-UTR of IGF-II mRNAs.

The insulin-like growth factor II mRNAs are targets for site-specific endonucleolytic cleavage in the 3'-UTR, which results in a very stable 3' cleavage product of 1.8 kb, consisting of 3'-UTR sequences and a poly(A) tail. The 5' cleavage product contains the coding region and is rapidly degraded. Thus, cleavage is thought to provide an additional way to control IGF-II protein synthesis. We had established that cleavage requires two widely separated sequence elements (I and II) in the 3'-UTR that form a stable duplex of 83 nucleotides. The cleavage-site itself is located in an internal loop preceded by two stable stem-loop structures. Furthermore, in a study which was based on RNA folding algorithms, we have shown that there are specific sequence and structural requirements for the cleavage reaction. Here, the functions of the different structural domains in cleavage were assessed by deletion/mutational analyses, and biochemical structure probing assays were performed to characterize better the RNA structures formed and to verify the computer folding predictions. The data suggest that the stem-loop domain contributes to maintain a highly specific c leavage-site by preventing the formation of alternative structures in the cleavage-site domain. Involvement of the nucleotides in the cleavage-site loop itself in non-Watson-Crick interactions may be important for providing a specific recognition surface for an endoribonuclease activity.

Construction and preliminary characterization of a library of "lethal" preterminal protein mutant adenoviruses.

Adenoviruses containing lethal in-frame insertion mutant alleles of the preterminal protein (pTP) gene were constructed with cell lines that express pTP. Thirty in-frame insertion mutant alleles, including 26 alleles previously characterized as lethal and 4 newly constructed mutant alleles, were introduced into the viral chromosome in place of the wild-type pTP gene. The viruses were tested for ability to form plaques at 37 degrees C in HeLa-pTP cells and at 32 degrees C and 39.5 degrees C in HeLa cells. Two of the newly constructed viruses exhibited temperature sensitivity for plaque formation, one virus did not form plaques in the absence of complementation, seven additional mutants exhibited a greater than 10-fold reduction in plaque formation in the absence of complementation, and another eight mutants exhibited stronger phenotypes than did previously characterized in-frame insertion mutants in the plaque assay. These mutant viruses offer promise for analysis of pTP functions.

The chum salmon IGF-II gene promoter is activated by hepatocyte nuclear factor 3beta.

IGF-II plays an important role in growth and development of vertebrates and is highly expressed in adult salmon liver. In the present study, we demonstrate that a liver-enriched transcription factor, hepatocyte nuclear factor 3beta (HNF-3beta), is an activator of the chum salmon IGF-II gene. Multiple binding sites for HNF-3beta were identified within the 5'-UTR using electrophoretic mobility shift assays and mutation of these sites prevents binding of HNF-3beta. In transient transfection assays it was shown that mutation of the HNF-3beta binding sites results in a substantial decrease of HNF-3beta-activated salmon IGF-II gene expression. This is the first identified transcription factor that is functionally involved in the regulation of fish IGF-II expression.

Growth hormone induces insulin-like growth factor-I gene transcription by a synergistic action of STAT5 and HNF-1alpha.

Salmon insulin-like growth factor-I (sIGF-I) expression is, as in mammals, induced by growth hormone (GH). To elucidate the mechanism by which GH stimulates the transcription of the IGF-I gene, we transiently transfected Hep3B cells expressing the rat GH receptor with a sIGF-I promoter-luciferase reporter construct. Activation of the construct by GH added to the medium of the transfected cells was observed when two specific transcription factors, STAT5 and HNF-1alpha, were simultaneously overexpressed in these cells. This finding demonstrates for the first time a GH-dependent activation of an IGF-I promoter construct in an immortalized laboratory cell line.

Dual role for transcription factor AP-2 in the regulation of the major fetal promoter P3 of the gene for human insulin-like growth factor II.

The human insulin-like growth factor II (IGF-II) gene contains four promoters that are differentially active during cell growth and development. Promoter 3 (P3) is the most active promoter in fetal and non-hepatic adult tissues. In addition to its expression during development, P3 is also the major promoter in many tumour tissues and IGF-II-expressing cell lines. Here we show that AP-2 has a dual function in P3 regulation in vivo as well as in vitro. In cells expressing low levels of endogenous AP-2, AP-2 overexpression activates P3, whereas P3 promoter activity is inhibited in cells containing abundant AP-2. Four potential AP-2-binding sites were identified in footprinting studies with recombinant AP-2. One of these AP-2-binding sites is located within the previously identified element P3-4 that contains two adjacent binding sites for IGF-II promoter-binding proteins IPBP3 and IPBP4/5. By applying binding competition assays and mutational analysis it is shown that AP-2 interferes with IPBP3 binding and transactivation in vivo as well as in vitro. Furthermore, AP-2 can bind additional elements in the proximal P3 promoter that also contribute to AP-2-mediated transactivation as shown by transient transfection assays. From these results we conclude that AP-2 is an important regulator in vivo and in vitro of IGF-II P3 activity.

Identification of RNA sequences and structures involved in site-specific cleavage of IGF-II mRNAs.

Insulin-like growth factor-II (IGF-II) mRNAs are subject to site-specific endonucleolytic cleavage in the 3' untranslated region (UTR), rendering an unstable 5' cleavage product containing the coding region and a very stable 3' cleavage product of 1.8 kb consisting of the 3'-UTR sequence and the poly(A) tail. Previously, it was established that two widely separated elements in the 3'-UTR (elements I and II), that can form a duplex structure, are necessary and sufficient for cleavage. To further investigate the sequence and secondary structure requirements for cleavage, we have introduced a number of mutations around the cleavage site and assayed their effects on cleavage. Several recognition determinants involved in the endonucleolytic cleavage of IGF-II mRNAs were identified. Mutational analysis around the cleavage site revealed that cleavage is sequence specific and that the cleavage site must be in a single-stranded conformation to allow efficient cleavage. In addition, we have identified an accessory protein that specifically interacts with a stem-loop structure located 133 to 73 nt upstream of the cleavage site.

The role of MAP kinase in TPA-mediated cell cycle arrest of human breast cancer cells.

In MCF7 breast cancer cells, mitogen-activated protein (MAP) kinase (i.e. Erk-1/2) is activated by the mitogen insulin, but also by the growth inhibiting agent TPA, though with very different kinetics. Insulin induces a relatively transient activation of Erk2 (<15 min), whereas TPA is able to induce a prolonged activation of Erk2 (>6 h). Expression of immediate-early genes of the c-fos and c-jun families, whose transcription and activation are regulated by MAP kinases, is differentially induced by insulin and TPA. Whereas insulin stimulates prolonged induction of c-jun, but not of junB mRNA, resulting in c-jun expression during the entire G1 period, the growth inhibitor TPA induces junB much longer than c-jun. Inhibition of the Erk2 pathway by PD98059, specific for the upstream MAP kinase kinase (MEK1), abolishes TPA-stimulated junB but not insulin-induced c-jun. In agreement with this, insulin readily stimulates Jun kinase (JNK), whereas TPA does not. Furthermore, insulin-induced pRB hyperphosphorylation at the G1-S transition and S-phase entry is insensitive to MAP kinase inhibition by PD98059. On the other hand, PD98059 reverts the inhibitory effect of TPA on cell cycle entry as well as on pRB hyperphosphorylation, indicating that Erk effectors function as inhibitors of proliferation in MCF7 cells.

Organization and expression of the chum salmon insulin-like growth factor II gene.

IGF-II plays an important role in growth and development of vertebrates. In the present study, the characterization of the first fish IGF-II gene, chum salmon IGF-II, is described. The sIGF-II gene consists of four exons, spanning a region of 9 kbp, that encode the 214 aa IGF-II precursor. While the amino acid sequences of fully processed IGF-II of salmon and mammalian species are very similar, the prepro-peptide sequence deviates extensively in the signal- and E-peptide domains. The transcription initiation site of the sIGF-II gene was localized within a 30 nt region employing RT-PCR. Using sIGF-II promoter-luciferase constructs it was demonstrated that the sIGF-II gene has a relatively strong promoter that contains tissue-specific regulatory elements.

A functional Sp1 binding site is essential for the activity of the adult liver-specific human insulin-like growth factor II promoter.

The human gene encoding insulin-like growth factor II contains four promoters (P1-P4) that are differentially activated in various tissues during development. Expression of insulin-like growth factor II in adult liver tissue is directed by P1, which is activated by liver-enriched members of the CCAAT/enhancer binding protein family of transcription factors. In the present report we show that the region around -48 relative to the transcription start site contains a high affinity Sp1 binding site. This was demonstrated by electrophoretic mobility shift assays using nuclear extracts from Hep3B hepatoma cells and with specific antibodies directed against Sp1. Competition electrophoretic mobility shift assays revealed that the Sp1 binding site of P1 and a consensus Sp1 binding site bind Sp1 with comparable efficiencies. Mutation of the Sp1 binding site results in an 85% decrease in P1 promoter activity in transient transfection assays using two different cell lines, COS-7 and Hep3B. Investigation of P1 mutants in which the spacing of the Sp1 binding site and the transcription start site was increased showed that the role of the Sp1 binding site in regulation of P1 is position dependent. Interestingly, the Sp1-responsive element cannot be exchanged by a functional TATA box. Activation of P1 by transactivators CCAAT/enhancer binding protein-beta and hepatocyte nuclear factor-3beta is strongly impaired after mutation of the Sp1 binding site. These results demonstrate that the specific presence of a binding site for the ubiquitously expressed transcription factor Sp1 is of eminent importance for efficient activation of P1 by liver-enriched transactivators.

Histamine-stimulated expression of insulin-like growth factors in human glioma cells.

Glioma tumour growth is associated with the expression of insulin-like growth factors I and II (IGFs) and of both type I and type II IGF receptors. It has also been shown that IGFs can stimulate proliferation of cultured glioma cells. We previously reported that histamine too can stimulate the growth of glioma cells in vitro. In this report, we study whether the histamine-induced growth of G47 glioma cells is mediated by the IGFs. We found that histamine stimulates the expression of both IGF-I and IGF-II mRNAs, as determined by a semiquantitative in situ hybridization analysis. Furthermore, incubation of G47 cells with histamine also induced cellular immunostaining for IGF-II. It could be shown that IGF-I-stimulated proliferation is inhibited by IGFBP-3, which decreases the availability of IGFs for binding to the IGF receptors, and by beta-galactosidase, which may decrease IGF binding to the type II IGF receptor, but is not inhibited by the anti-type I IGF receptor monoclonal antibody alphaIR3. However, neither IGFBP-3 nor beta-galactosidase nor alphaIR3 inhibited the histamine-induced proliferation. These results show that the growth-stimulatory effect of histamine is accompanied by the induction of IGFs. This histamine-induced growth stimulation is not mediated by activation of cell surface IGF receptors, although intracrine activation of type II IGF receptors may be involved.

Expression of insulin-like growth factors (IGFs), their receptors and IGF binding protein-3 in normal, benign and malignant smooth muscle tissues.

To assess the role of insulin-like growth factors (IGFs) in growth and transformation of normal (myometrium) and tumorous smooth muscle cell (SMC) tissues, in situ hybridization (ISH) analysis for insulin-like growth factor I and II (IGF-I and IGF-II) mRNAs was combined with detection of IGF peptides, their receptors and IGF binding protein-3 (IGFBP-3). mRNAs for both IGFs were detected in smooth muscle cells in normal, benign and malignant SMC tissues, together with the IGF peptides, both IGF receptors and IGFBP-3. This suggests an autocrine role for both IGFs. Leiomyomas had higher IGF-I peptide levels and higher levels of type I IGF receptors than myometrium, supporting the idea that IGFs play a role in the growth and transformation of these tumours. Low-grade leiomyosarcomas contained more IGF-II mRNAs than myometrium and leiomyoma, fewer type II IGF/mannose 6-phosphate receptors and less IGFBP-3 than myometrium and, in addition, fewer IGF-I mRNAs and type I IGF receptors than leiomyoma. Intermediate- and high-grade leiomyosarcomas had intermediate levels of IGF-II mRNAs and peptide, ranging between those in myometrium and low-grade leiomyosarcomas. Thus, growth and transformation of leiomyosarcomas may be regulated by IGF-II, although more markedly in low-grade than in high-grade leiomyosarcomas. In conclusion, the various categories of SMC tissues are associated with a distinct expression pattern of the IGF system. This suggests that each category of SMC tumours arises as a distinct entity and that there is no progression of transformation in these tissues.

Mitogenic signaling of insulin-like growth factor I in MCF-7 human breast cancer cells requires phosphatidylinositol 3-kinase and is independent of mitogen-activated protein kinase.

Addition of insulin-like growth factor I (IGF-I) to quiescent breast tumor-derived MCF-7 cells causes stimulation of cyclin D1 synthesis, hyperphosphorylation of the retinoblastoma protein pRb, DNA synthesis, and cell division. All of these effects are independent of the mitogen-activated protein kinase (MAPK) pathway since none of them is blocked by PD098059, the specific inhibitor of the MAPK activating kinase MEK1. This observation is consistent with the finding that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a strong inducer of MAPK activity in MCF-7 cells, effectively inhibits proliferation. The anti-proliferative effect of TPA in these cells may be accounted for, at least in part, by the MAPK-dependent stimulation of the synthesis of p21(WAF1/CIP1), an inhibitor of cyclin/cyclin-dependent kinase complexes. In contrast, all of the observed stimulatory effects of IGF-I on cell cycle progression, cyclin D1 synthesis, and pRb hyperphosphorylation were blocked by the specific phosphatidylinositol 3-kinase inhibitor LY294002, suggesting that phosphatidylinositol 3-kinase activity but not MAPK activity is required for transduction of the mitogenic IGF-I signal in MCF-7 cells.

Identification of a key regulatory element for the basal activity of the human insulin-like growth factor II gene promoter P3.

Transcription of the human insulin-like growth factor II (IGF-II) gene is under the control of four promoters (P1-P4) that are differentially active during growth and development. Promoter 3 (P3) is the most active promoter during fetal development as well as in most adult tissues. P3 is also the most active promoter in tumour tissues and cell lines expressing IGF-II. Transient transfections of HeLa and Hep3B cells with truncated promoter constructs revealed that the region between -289 and -183 relative to the transcription start site supports basal promoter activity in both cell lines. Footprint experiments showed that the region between positions -192 and -172 (P3-4) is the only element bound by nuclear proteins. P3-4 is bound by five proteins, of which three proteins (proteins 3, 4 and 5) bind specifically and are expressed at the same levels in HeLa and Hep3B cells. Electrophoretic mobility shift assays and differential footprint experiments revealed the presence of two protein-binding regions within the P3-4 element. Proteins 4 and 5 bind box A (-193 to -188), whereas box B (-183 to -172) is bound by protein 3. From transcription experiments in vitro it can be concluded that Box A is essential for P3 activity. Box A is part of a region 11 dG residues long and is protected by proteins 4 and 5 that bind a contiguous set of six dG residues. DNA-binding of proteins 4 and 5 to box A requires the presence of Zn2+ ions. Thus structural and functional analysis reveals that the P3-4 element is a key regulatory element of P3 that contains two separate binding sites for proteins essential for the basal activity of IGF-II P3.

The hepatocyte nuclear factor 3beta stimulates the transcription of the human insulin-like growth factor I gene in a direct and indirect manner.

Promoter 1 (P1) of the human insulin-like growth factor I (IGF-I) gene is most active in adult liver. In this study we show that HNF-3beta, a member of the winged helix protein family of liver-enriched transcription factors, has a strong stimulatory effect on the activity of P1. Transient transfection experiments in combination with bandshift and DNase I footprinting analysis revealed the presence of two HNF-3 binding sites in the proximal promoter region of P1. Both binding sites, which are well conserved in evolution, are required for maximal transactivation. Studies employing HNF-3 mutant constructs indicated that IGF-I expression is also regulated indirectly by HNF-3beta as a consequence of enhanced expression of HNF-1alpha. This liver-enriched transcription factor has previously been shown to transactivate P1. Thus, HNF-3beta regulates the expression of the human IGF-I gene via two distinct mechanisms.

Modulation of insulin-like growth factor (IGF) action by IGF-binding proteins in normal, benign, and malignant smooth muscle tissues.

The insulin-like growth factor (IGF) system is involved in the growth of uterine leiomyomas (L), as these tumors have higher IGF-II messenger ribonucleic acid levels, type I IGF receptor levels, and IGF-I peptide concentrations than myometrium (M). Furthermore, cultured L smooth muscle cells (SMC) respond with greater efficiency to IGF-I than M SMC. Here we investigate a possible modulating role of the binding proteins for the IGFs (IGFBPs) on the actions of IGFs. IGFBP-3 is the most predominant IGFBP in conditioned medium from SMC, with levels ranging from 13-288 ng/mL. Incubation of SMC cultures with IGF-I and the IGF-I analogs long-R3IGF-I and des(1-3)-IGF-I, which have decreased affinity for IGFBPs, revealed a facilitating effect of IGFBPs on the growth-stimulating activity of a high concentration of IGF-I in cell lines with high IGFBP-3 levels. Both a decreased level of IGFBP-3 and a low concentration of the growth factors added were a disadvantage for the facilitating effect. In M and L tissue sections, IGFBP-3 was found exclusively bound to the constituting cells, not in the extracellular matrix. This suggests that a negative modulating role of IGFBP-3 due to sequestration of IGF-I, as occurs in culture medium, is less relevant in vivo. In leiomyosarcoma sections, IGFBP-3 levels are decreased, indicating a decreasing, role for this binding protein in malignant smooth muscle tissues.

The liver-specific promoter of the human insulin-like growth factor-II gene contains two negative regulatory elements.

The adult liver-specific IGF-II promoter P1 is activated by CCAAT/Enhancer Binding Proteins alpha and beta. Here we present evidence that promoter P1, in addition to positively regulating elements, contains two elements of 67 nucleotides that form an inverted repeat (IR) and suppress P1 activity. The two IR elements are specifically bound by a protein (inverted repeat binding factor, IRBF). The amounts of IRBF in various cell lines correlate with the levels of suppression of P1 activity, suggesting that this factor is responsible for the suppression of P1 mediated by the IR elements.

Growth-condition-dependent regulation of insulin-like growth factor II mRNA stability.

Insulin-like growth factor II (IGF-II) is synthesized in many tissues, but the main site of production is the liver. In this paper we show that IGF-II mRNA levels are dependent on the growth conditions of the cells. In Hep3B cells, serum deprivation leads to a marked increase in IGF-II mRNA levels. Serum stimulation of starved Hep3B cells induces a decrease in the amount of IGF-II mRNA, which is not caused by a change in promoter activity. IGF-II mRNAs are subject to endonucleolytic cleavage, a process that requires two widely separated elements in the 3' untranslated region of the mRNA. Specific regions of these elements can form a stable stem structure which is involved in the formation of RNA-protein complexes. By employing electrophoretic mobility shift assays, two complexes have been identified in cytoplasmic extracts of Hep3B cells. The formation of these complexes is related to the growth conditions of the cells and is correlated with the regulation of IGF-II mRNA levels. Our data suggest that, depending on whether serum is present or absent, a transition from one complex to the other occurs. A decrease in the IGF-II mRNA level is also observed when IGF-I or IGF-II is added to serum-deprived Hep3B cells, possibly providing a feedback mechanism for IGF-II production. The serum-induced degradation of IGF-II mRNAs does not require de novo protein synthesis, and is abolished by rapamycin, an inhibitor of p70 S6 kinase.