The Activity of Differentiation Factors Induces Apoptosis in Polyomavirus Large T-Expressing Myoblasts

It is commonly accepted that pathways that regulate proliferation/differentiation processes, if altered in their normal interplay, can lead to the induction of programmed cell death. In a previous work we reported that Polyoma virus Large Tumor antigen (PyLT) interferes with in vitro terminal differentiation of skeletal myoblasts by binding and inactivating the retinoblastoma antioncogene product. This inhibition occurs after the activation of some early steps of the myogenic program. In the present work we report that myoblasts expressing wild-type PyLT, when subjected to differentiation stimuli, undergo cell death and that this cell death can be defined as apoptosis. Apoptosis in PyLT-expressing myoblasts starts after growth factors removal, is promoted by cell confluence, and is temporally correlated with the expression of early markers of myogenic differentiation. The block of the initial events of myogenesis by transforming growth factor β or basic fibroblast growth factor prevents PyLT-induced apoptosis, while the acceleration of this process by the overexpression of the muscle-regulatory factor MyoD further increases cell death in this system. MyoD can induce PyLT-expressing myoblasts to accumulate RB, p21, and muscle- specific genes but is unable to induce G00 arrest. Several markers of different phases of the cell cycle, such as cyclin A, cdk-2, and cdc-2, fail to be down-regulated, indicating the occurrence of cell cycle progression. It has been frequently suggested that apoptosis can result from an unbalanced cell cycle progression in the presence of a contrasting signal, such as growth factor deprivation. Our data involve differentiation pathways, as a further contrasting signal, in the generation of this conflict during myoblast cell apoptosis.

Activation of Utrophin Promoter by Heregulin via the ets-related Transcription Factor Complex GA-binding Protein α/β

Utrophin/dystrophin-related protein is the autosomal homologue of the chromosome X-encoded dystrophin protein. In adult skeletal muscle, utrophin is highly enriched at the neuromuscular junction. However, the molecular mechanisms underlying regulation of utrophin gene expression are yet to be defined. Here we demonstrate that the growth factor heregulin increases de novo utrophin transcription in muscle cell cultures. Using mutant reporter constructs of the utrophin promoter, we define the N-box region of the promoter as critical for heregulin-mediated activation. Using this region of the utrophin promoter for DNA affinity purification, immunoblots, in vitro kinase assays, electrophoretic mobility shift assays, and in vitro expression in cultured muscle cells, we demonstrate that ets-related GA-binding protein α/β transcription factors are activators of the utrophin promoter. Taken together, these results suggest that the GA-binding protein α/β complex of transcription factors binds and activates the utrophin promoter in response to heregulin-activated extracellular signal–regulated kinase in muscle cell cultures. These findings suggest methods for achieving utrophin up-regulation in Duchenne’s muscular dystrophy as well as mechanisms by which neurite-derived growth factors such as heregulin may influence the regulation of utrophin gene expression and subsequent enrichment at the neuromuscular junction of skeletal muscle.

Transcriptional Regulation of Fibroblast Growth Factor-2 Expression in Human Astrocytes: Implications for Cell Plasticity

Induction of the fibroblast growth factor-2 (FGF-2) gene and the consequent accumulation of FGF-2 in the nucleus are operative events in mitotic activation and hypertrophy of human astrocytes. In the brain, these events are associated with cellular degeneration and may reflect release of the FGF-2 gene from cell contact inhibition. We used cultures of human astrocytes to examine whether expression of FGF-2 is also controlled by soluble growth factors. Treatment of subconfluent astrocytes with interleukin-1β, epidermal or platelet-derived growth factors, 18-kDa FGF-2, or serum or direct stimulation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate or adenylate cyclase with forskolin increased the levels of 18-, 22-, and 24-kDa FGF-2 isoforms and FGF-2 mRNA. Transfection of FGF-2 promoter–luciferase constructs identified a unique −555/−513 bp growth factor-responsive element (GFRE) that confers high basal promoter activity and activation by growth factors to a downstream promoter region. It also identified a separate region (−624/−556 bp) essential for PKC and cAMP stimulation. DNA–protein binding assays indicated that novel cis-acting elements and trans-acting factors mediate activation of the FGF-2 gene. Southwestern analysis identified 40-, 50-, 60-, and 100-kDa GFRE-binding proteins and 165-, 112-, and 90-kDa proteins that interacted with the PKC/cAMP-responsive region. The GFRE and the element essential for PKC and cAMP stimulation overlap with the region that mediates cell contact inhibition of the FGF-2 promoter. The results show a two-stage regulation of the FGF-2 gene: 1) an initial induction by reduced cell contact, and 2) further activation by growth factors or the PKC-signaling pathway. The hierarchic regulation of the FGF-2 gene promoter by cell density and growth factors or PKC reflects a two-stage activation of protein binding to the GFRE and to the PKC/cAMP-responsive region, respectively.

Stimulation of β1-Integrin Function by Epidermal Growth Factor and Heregulin-β Has Distinct Requirements for erbB2 but a Similar Dependence on Phosphoinositide 3-OH Kinase

Integrins and growth factor receptors are important participants in cellular adhesion and migration. The EGF receptor (EGFR) family of tyrosine kinases and the β1-integrin adhesion receptors are of particular interest, given the implication for their involvement in the initiation and progression of tumorigenesis. We used adhesion and chemotaxis assays to further elucidate the relationship between these two families of transmembrane signaling molecules. Specifically, we examined integrin-mediated adhesive and migratory characteristics of the metastatic breast carcinoma cell line MDA-MB-435 in response to stimulation with growth factors that bind to and activate the EGFR or erbB3 in these cells. Although ligand engagement of the EGFR stimulated modest β1-dependent increases in cell adhesion and motility, heregulin-β (HRGβ) binding to the erbB3 receptor initiated rapid and potent induction of breast carcinoma cell adhesion and migration and required dimerization of erbB3 with erbB2. Pharmacologic inhibitors of phosphoinositide 3-OH kinase (PI 3-K) or transient expression of dominant negative forms of PI 3-K inhibited both EGF- and HRGβ-mediated adhesion and potently blocked HRGβ- and EGF-induced cell motility. Our results illustrate the critical role of PI 3-K activity in signaling pathways initiated by the EGFR or erbB3 to up-regulate β1-integrin function.

Involvement of protein kinase A in fibroblast growth factor-2-activated transcription

Polypeptide growth factors activate common signal transduction pathways, yet they can induce transcription of different target genes. The mechanisms that control this specificity are not completely understood. Recently, we have described a fibroblast growth factor (FGF)-inducible response element, FiRE, on the syndecan-1 gene. In NIH 3T3 cells, the FiRE is activated by FGF-2 but not by several other growth factors, such as platelet-derived growth factor or epidermal growth factor, suggesting that FGF-2 activates signaling pathways that diverge from pathways activated by other growth factors. In this paper, we report that the activation of FiRE by FGF-2 requires protein kinase A (PKA) in NIH 3T3 cells. The PKA-specific inhibitor H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) blocked the FGF-2-induced activation of FiRE, the transcription of the syndecan-1 gene, and cell proliferation. Also, expression of a dominant-negative form of PKA inhibited the FGF-2-induced FiRE activation and the transcription of the syndecan-1 gene. The binding of activator protein-1 transcription-factor complexes, required for the activation of FiRE, was blocked by inhibition of PKA activity before FGF-2 treatment. In accordance with the growth factor specificity of FiRE, the activity of PKA was stimulated by FGF-2 but not by platelet-derived growth factor or epidermal growth factor. Furthermore, a portion of the PKA catalytic subunit pool was translocated to the nucleus by FGF-2. Noticeably, the total cellular cAMP concentration was not affected by FGF-2 stimulus. We propose that the FGF-2-selective transcriptional activation through FiRE is caused by the ability of FGF-2 to control PKA activity.

CYR61, a product of a growth factor-inducible immediate early gene, promotes angiogenesis and tumor growth

CYR61 is a secreted, cysteine-rich, heparin-binding protein encoded by a growth factor-inducible immediate–early gene. Acting as an extracellular, matrix-associated signaling molecule, CYR61 promotes the adhesion of endothelial cells through interaction with the integrin αVβ3 and augments growth factor-induced DNA synthesis in the same cell type. In this study, we show that purified CYR61 stimulates directed migration of human microvascular endothelial cells in culture through an αVβ3-dependent pathway and induces neovascularization in rat corneas. Both the chemotactic and angiogenic activities of CYR61 can be blocked by specific anti-CYR61 antibodies. Whereas most human tumor-derived cell lines tested express CYR61, the gastric adenocarcinoma cell line RF-1 does not. Expression of the CYR61 cDNA under the regulation of a constitutive promoter in RF-1 cells significantly enhances the tumorigenicity of these cells as measured by growth in immunodeficient mice, resulting in tumors that are larger and more vascularized than those produced by control RF-1 cells. Taken together, these results identify CYR61 as an angiogenic inducer that can promote tumor growth and vascularization; the results also suggest potential roles for CYR61 in physiologic and pathologic neovascularization.

Pituitary Ets-1 and GABP bind to the growth factor regulatory sites of the rat prolactin promoter

Ets factors play a critical role in oncogenic Ras- and growth factor-mediated regulation of the proximal rat prolactin (rPRL) promoter in pituitary cells. The rPRL promoter contains two key functional Ets binding sites (EBS): a composite EBS/Pit-1 element located at –212 and an EBS that co-localizes with the basal transcription element (BTE, or A-site) located at –96. Oncogenic Ras exclusively signals to the –212 site, which we have named the Ras response element (RRE); whereas the response of multiple growth factors (FGFs, EGF, IGF, insulin and TRH) maps to both EBSs. Although Ets-1 and GA binding protein (GABP) have been implicated in the Ras and insulin responses, respectively, the precise identity of the pituitary Ets factors that specifically bind to the RRE and BTE sites remains unknown. In order to identify the Ets factor(s) present in GH4 and GH3 nuclear extracts (GH4NE and GH3NE) that bind to the EBSs contained in the RRE and BTE, we used EBS-RRE and BTE oligonucleotides in electrophoretic mobility shift assays (EMSAs), antibody supershift assays, western blot analysis of partially purified fractions and UV-crosslinking studies. EMSAs, using either the BTE or EBS-RRE probes, identified a specific protein–DNA complex, designated complex A, which contains an Ets factor as determined by oligonucleotide competition studies. Using western blot analysis of GH3 nuclear proteins that bind to heparin–Sepharose, we have shown that Ets-1 and GABP, which are MAP kinase substrates, co-purify with complex A, and supershift analysis with specific antisera revealed that complex A contains Ets-1, GABPα and GABPβ1. In addition, we show that recombinant full-length Ets-1 binds equivalently to BTE and EBS-RRE probes, while recombinant GABPα/β preferentially binds to the BTE probe. Furthermore, comparing the DNA binding of GH4NE containing both Ets-1 and GABP and HeLa nuclear extracts devoid of Ets-1 but containing GABP, we were able to show that the EBS-RRE preferentially binds Ets-1, while the BTE binds both GABP and Ets-1. Finally, UV-crosslinking experiments with radiolabeled EBS-RRE and BTE oligonucleotides showed that these probes specifically bind to a protein of ∼64 kDa, which is consistent with binding to Ets-1 (54 kDa) and/or the DNA binding subunit of GABP, GABPα (57 kDa). These studies show that endogenous, pituitary-derived GABP and Ets-1 bind to the BTE, whereas Ets-1 preferentially binds to the EBS-RRE. Taken together, these data provide important insights into the mechanisms by which the combination of distinct Ets members and EBSs transduce differential growth factor responses.

Alternative splicing of agrin regulates its binding to heparin alpha-dystroglycan, and the cell surface.

Agrin is a basal lamina molecule that directs key events in postsynaptic differentiation, most notably the aggregation of acetylcholine receptors (AChRs) on the muscle cell surface. Agrin's AChR clustering activity is regulated by alternative mRNA splicing. Agrin splice forms having inserts at two sites (y and z) in the C-terminal region are highly active, but isoforms lacking these inserts are weakly active. The biochemical consequences of this alternative splicing are unknown. Here, the binding of four recombinant agrin isoforms to heparin, to alpha-dystroglycan (a component of an agrin receptor), and to myoblasts was tested. The presence of a four-amino acid insert at the y site is necessary and sufficient to confer heparin binding ability to agrin. Moreover, the binding of agrin to alpha-dystroglycan is inhibited by heparin when this insert is present. Agrin binding to the cell surface showed analogous properties: heparin inhibits the binding of only those agrin isoforms containing this four-amino acid insert. The results show that alternative splicing of agrin regulates its binding to heparin and suggest that agrin's interaction with alpha-dystroglycan may be modulated by cell surface glycosaminoglycans in an isoform-dependent manner.

Insulin-like growth factor II mediates epidermal growth factor-induced mitogenesis in cervical cancer cells.

There is increasing evidence that activation of the insulin-like growth factor I (IGF-I) receptor plays a major role in the control of cellular proliferation of many cell types. We studied the mitogenic effects of IGF-I, IGF-II, and epidermal growth factor (EGF) on growth-arrested HT-3 cells, a human cervical cancer cell line. All three growth factors promoted dose-dependent increases in cell proliferation. In untransformed cells, EGF usually requires stimulation by a "progression" factor such as IGF-I, IGF-II, or insulin (in supraphysiologic concentrations) in order to exert a mitogenic effect. Accordingly, we investigated whether an autocrine pathway involving IGF-I or IGF-II participated in the EGF-induced mitogenesis of HT-3 cells. With the RNase protection assay, IGF-I mRNA was not detected. However, IGF-II mRNA increased in a time-dependent manner following EGF stimulation. The EGF-induced mitogenesis was abrogated in a dose-dependent manner by IGF-binding protein 5 (IGFBP-5), which binds to IGF-II and neutralizes it. An antisense oligonucleotide to IGF-II also inhibited the proliferative response to EGF. In addition, prolonged, but not short-term, stimulation with EGF resulted in autophosphorylation of the IGF-I receptor, and coincubations with both EGF and IGFBP-5 attenuated this effect. These data demonstrate that autocrine secretion of IGF-II in HT-3 cervical cancer cells can participate in EGF-induced mitogenesis and suggest that autocrine signals involving the IGF-I receptor occur "downstream" of competence growth factor receptors such as the EGF receptor.

Hypoxia-mediated induction of acidic/basic fibroblast growth factor and platelet-derived growth factor in mononuclear phagocytes stimulates growth of hypoxic endothelial cells.

Wound repair and tumor vascularization depend upon blood vessel growth into hypoxic tissue. Although hypoxia slows endothelial cell (EC) proliferation and suppresses EC basic fibroblast growth factor (bFGF) expression, we report that macrophages (MPs) exposed to PO2 approximately 12-14 torr (1 torr = 133.3 Pa) synthesize and release in a time-dependent manner platelet-derived growth factor (PDGF) and acidic/basic FGFs (a/bFGFs), which stimulate the growth of hypoxic ECs. Chromatography of hypoxic MP-conditioned medium on immobilized heparin with an ascending NaCl gradient resolved three peaks of mitogenic activity: activity of the first peak was neutralized by antibody to PDGF; activity of the second peak was neutralized by antibody to aFGF; and activity of the third peak was neutralized by antibody to bFGF. Metabolically labeled lysates and supernatants from MPs exposed to hypoxia showed increased synthesis and release of immunoprecipitable PDGF and a/bFGF in the absence of changes in cell viability. Possible involvement of a heme-containing oxygen sensor in MP elaboration of growth factors was suggested by the induction of bFGF and PDGF by normoxic MPs exposed to nickel or cobalt, although metabolic inhibitors such as sodium azide were without effect. These results suggest a paracrine model in which hypoxia stimulates MP release of PDGF and a/bFGF, inducing EC proliferation and potentially promoting angiogenesis in hypoxic environments.

Induction of cell proliferation in mammalian inner-ear sensory epithelia by transforming growth factor alpha and epidermal growth factor.

Regenerative proliferation occurs in the inner-ear sensory epithelial of warm-blooded vertebrates after insult. To determine how this proliferation is controlled in the mature mammalian inner ear, several growth factors were tested for effects on progenitor-cell division in cultured mouse vestibular sensory epithelia. Cell proliferation was induced in the sensory epithelium by transforming growth factor alpha (TGF-alpha) in a dose-dependent manner. Proliferation was also induced by epidermal growth factor (EGF) when supplemented with insulin, but not EGF alone. These observations suggest that stimulation of the EGF receptors by TGF-alpha binding, or EGF (plus insulin) binding, stimulates cell proliferation in the mature mammalian vestibular sensory epithelium.

Growth hormone as a cytokine

1. The growth hormone (GH) receptor was the first of the class 1 cytokine receptors to be cloned. It shares a number of structural characteristics with other family members and common signalling mechanisms based on common usage of the Janus kinase 2 (JAK2). 2. Growth hormone receptor activation is initiated by GH-induced homodimerization of receptor molecules. This has enabled the creation of specific hormone antagonists that block receptor dimerization. 3. The details of the transcription factors used by the activated receptor are being revealed as a result of promoter analyses and electrophoretic mobility gelshift analysis. 4. Growth hormone receptors are widespread and their discovery in certain tissues has led to the assignment of new physiological roles for GH, Some of these involve local or paracrine roles for GH, as befits its cytokine status. 5. Four examples of such novel roles are discussed, These are: (i) the brain GH axis; (ii) GH and the vitamin B-12 axis; (iii) GH in early pre-implantation development; and (iv) GH in development of the tooth. 6. We propose that the view that GH acts through the intermediacy of insulin-like growth factor-1 is simplistic; rather, GH acts to induce an array of growth factors and their receptors and the composition of this array varies with tissue type and, probably, stage of development.

Factors important to the secretion and matrix deposition of tissue transglutaminase

Data suggest that for TG2 to be secreted, an intact N-terminal FN binding site (for which TG2 has high affinity) is required, however interaction of TG2 with its high affinity binding partners presents both in the intracellular and extracellular space as well as with specific cell surface receptors may also be involved in this process. Using a site-directed mutagenesis approach, the effects of specific mutations of TG2 on its translocation to the cell surface and secretion into the ECM have been investigated. Mutations include those affecting FN binding (FN1), HSPGs binding (HS1, HS2) GTP/GDP binding site (GTP1, 2) as well as N-terminal and C-terminal domains (TG2 deletion mutants N, and C). By performing transglutaminase activity assays, cell surface protein biotinylation and verifying distribution of TG2 mutants in the ECM we demonstrated that one of the potential heparan sulfate binding site mutants (HS2 mutant) is secreted at the cell surface in a much reduced manner and is less deposited into the ECM than the HS1 mutant. The HS2 mutant showed a low affinity for binding to a heparin sepharose column demonstrating this mutation site may be a potential heparan binding site of TG2. Analogous peptides to this site were shown to have some efficiency in the inhibition of the binding of the FN-TG2 complex to cell surface heparan sulfates in a cell adhesion assay indicating the peptide to be representative of the novel heparin binding site within TG2. The GTP binding site mutants GTP1 and GTP2 exhibited low specific activity however, GTP2 showed more secretion to the cell surface in comparison to GTP1. The FN1 binding mutant did not greatly affect TG2 activity nor did it alter TG2 secretion at the cell surface and deposition into the ECM indicating that fibronectin binding at this site on the enzyme is not an important factor. Interestingly an intact N-terminus (?1-15) appeared to be essential for enzyme externalisation. Removal of the first 15 amino acids (N-terminal mutant) abolished TG2 secretion to the cell surface as well as deposition into the ECM. In addition it reduced the enzymes affinity for binding to heparin. In contrast, deletion of the C-terminal TG2 domain (?594-687) increased enzyme secretion to the cell surface. Consistent with the data presented in this thesis we speculate that TG2 must fulfill two requirements to be successfully secreted from cells. The findings indicate that the closed conformation of the enzyme as well as intact N-terminal tail and a novel HS binding site within the TG2 molecule are key elements for the enzyme’s localisation at the cell surface and its deposition into the extracellular matrix. The importance of understanding the interactions between TG2, heparan sulfates and other TG2 binding partners at the cell surface could have an impact on the design of novel strategies for enzyme inhibition which could be important in the control of extracellular TG2 related diseases.

Hormônio do crescimento e exercício físico: considerações atuais

Embora o hormônio do crescimento (GH) seja um dos hormônios mais estudados, vários de seus aspectos fisiológicos ainda não estão integralmente esclarecidos, incluindo sua relação com o exercício físico. Estudos mais recentes têm aumentado o conhecimento a respeito dos mecanismos de ação do GH, podendo ser divididos em: 1) ações diretas, mediadas pela rede de sinalizações intracelulares, desencadeadas pela ligação do GH ao seu receptor na membrana plasmática; e 2) ações indiretas, mediadas principalmente pela regulação da síntese dos fatores de crescimento semelhantes à insulina (IGF). Tem sido demonstrado que o exercício físico é um potente estimulador da liberação do GH. A magnitude deste aumento sofre influência de diversos fatores, em especial, da intensidade e do volume do exercício, além do estado de treinamento. Atletas, normalmente, apresentam menor liberação de GH induzida pelo exercício que indivíduos sedentários ou pouco treinados. Evidências experimentais demonstram que o GH: 1) favorece a mobilização de ácidos graxos livres do tecido adiposo para geração de energia; 2) aumenta a capacidade de oxidação de gordura e 3) aumenta o gasto energético.

Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets

Background: Melanoma progression occurs through three major stages: radial growth phase (RGP), confined to the epidermis; vertical growth phase (VGP), when the tumor has invaded into the dermis; and metastasis. In this work, we used suppression subtractive hybridization (SSH) to investigate the molecular signature of melanoma progression, by comparing a group of metastatic cell lines with an RGP-like cell line showing characteristics of early neoplastic lesions including expression of the metastasis suppressor KISS1, lack of alpha v beta 3-integrin and low levels of RHOC. Methods: Two subtracted cDNA collections were obtained, one (RGP library) by subtracting the RGP cell line (WM1552C) cDNA from a cDNA pool from four metastatic cell lines (WM9, WM852, 1205Lu and WM1617), and the other (Met library) by the reverse subtraction. Clones were sequenced and annotated, and expression validation was done by Northern blot and RT-PCR. Gene Ontology annotation and searches in large-scale melanoma expression studies were done for the genes identified. Results: We identified 367 clones from the RGP library and 386 from the Met library, of which 351 and 368, respectively, match human mRNA sequences, representing 288 and 217 annotated genes. We confirmed the differential expression of all genes selected for validation. In the Met library, we found an enrichment of genes in the growth factors/receptor, adhesion and motility categories whereas in the RGP library, enriched categories were nucleotide biosynthesis, DNA packing/repair, and macromolecular/vesicular trafficking. Interestingly, 19% of the genes from the RGP library map to chromosome 1 against 4% of the ones from Met library. Conclusion: This study identifies two populations of genes differentially expressed between melanoma cell lines from two tumor stages and suggests that these sets of genes represent profiles of less aggressive versus metastatic melanomas. A search for expression profiles of melanoma in available expression study databases allowed us to point to a great potential of involvement in tumor progression for several of the genes identified here. A few sequences obtained here may also contribute to extend annotated mRNAs or to the identification of novel transcripts.