Biomedical subjects

Joseph A Martial

Publications and source records attributed to Joseph A Martial.

14 recordsLinked to original sources

PubMed2006

Prolactin/growth hormone-derived antiangiogenic peptides highlight a potential role of tilted peptides in angiogenesis.

Angiogenesis is a crucial step in many pathologies, including tumor growth and metastasis. Here, we show that tilted peptides exert antiangiogenic activity. Tilted (or oblique-oriented) peptides are short peptides known to destabilize membranes and lipid cores and characterized by an asymmetric distribution of hydrophobic residues along the axis when helical. We have previously shown that 16-kDa fragments of the human prolactin/growth hormone (PRL/GH) family members are potent angiogenesis inhibitors. Here, we demonstrate that all these fragments possess a 14-aa sequence having the characteristics of a tilted peptide. The tilted peptides of human prolactin and human growth hormone induce endothelial cell apoptosis, inhibit endothelial cell proliferation, and inhibit capillary formation both in vitro and in vivo. These antiangiogenic effects are abolished when the peptides' hydrophobicity gradient is altered by mutation. We further demonstrate that the well known tilted peptides of simian immunodeficiency virus gp32 and Alzheimer's beta-amyloid peptide are also angiogenesis inhibitors. Taken together, these results point to a potential new role for tilted peptides in regulating angiogenesis.

Alzheimer Disease↗

PubMed2005

Expression of the somatolactin beta gene during zebrafish embryonic development.

Somatolactin (Sl) is a pituitary hormone closely related to prolactin (Prl) and growth hormone that was until now only found in various fish species. We isolated the cDNA coding for zebrafish Slbeta and we identified the gene encoding this hormone. We also obtained a 1kb genomic fragment corresponding to the slbeta upstream promoter region. Furthermore, the slbeta expression pattern was examined during zebrafish embryogenesis using whole-mount in situ hybridization. Slbeta mRNA is first detected in a single cell at the anterior border of the neural plate starting at 23h post fertilization (hpf). Slbeta-expressing cells also express the transcription factor pit1 and are located close to prl-expressing cells. Using combined fluorescent in situ hybridization, we show that slbeta- and prl-expressing cells are clearly distinct at 29 hpf. Starting at 30 hpf, the number of slbeta positive cells increases and their location becomes more clearly distinct from lactotrope cells, in a more posterior position. At later stages (48 hpf), slbeta expression was observed posterior to growth hormone expression, again in a distinct cell type. We show that zebrafish mutants aal, as well as mutants in the pit1 gene, are deficient in slbeta expression. In conclusion, slbeta expression defines a new, additional cell type in zebrafish pituitary that depends on pit1 and aal for its differentiation.

Animals↗

PubMed2005

sox4b is a key player of pancreatic alpha cell differentiation in zebrafish.

Pancreas development relies on a network of transcription factors belonging mainly to the Homeodomain and basic Helix-Loop-Helix families. We show in this study that, in zebrafish, sox4, a member of the SRY-like HMG-box (SOX) family, is required for proper endocrine cell differentiation. We found that two genes orthologous to mammalian Sox4 are present in zebrafish and that only one of them, sox4b, is strongly expressed in the pancreatic anlage. Transcripts of sox4b were detected in mid-trunk endoderm from the 5-somite stage, well before the onset of expression of the early pancreatic gene pdx-1. Furthermore, by fluorescent double in situ hybridization, we found that expression of sox4b is mostly restricted to precursors of the endocrine compartment. This expression is not maintained in differentiated cells although transient expression can be detected in alpha cells and some beta cells. That sox4b-expressing cells belong to the endocrine lineage is further illustrated by their absence from the pancreata of slow-muscle-omitted mutant embryos, which specifically lack all early endocrine markers while retaining expression of exocrine markers. The involvement of sox4b in cell differentiation is suggested firstly by its up-regulation in mind bomb mutant embryos displaying accelerated pancreatic cell differentiation. In addition, sox4b knock-down leads to a drastic reduction in glucagon expression, while other pancreatic markers including insulin, somatostatin, and trypsin are not significantly affected. This disruption of alpha cell differentiation is due to down-regulation of the homeobox arx gene specifically in the pancreas. Taken together, these data demonstrate that, in zebrafish, sox4b is expressed transiently during endocrine cell differentiation and plays a crucial role in the generation of alpha endocrine cells.

Amino Acid Sequence↗

PubMed2005

Solution structure of human prolactin.

We report the solution structure of human prolactin determined by NMR spectroscopy. Our result is a significant improvement over a previous structure in terms of number and distribution of distance restraints, regularity of secondary structure, and potential energy. More significantly, the structure is sufficiently different that it leads to different conclusions regarding the mechanism of receptor activation and initiation of signal transduction. Here, we compare the structure of unbound prolactin to structures of both the homologue ovine placental lactogen and growth hormone. The structures of unbound and receptor bound prolactin/placental lactogen are similar and no noteworthy structural changes occur upon receptor binding. The observation of enhanced binding at the second receptor site when the first site is occupied has been widely interpreted to indicate conformational change induced by binding the first receptor. However, our results indicate that this enhanced binding at the second site could be due to receptor-receptor interactions or some other free energy sources rather than conformational change in the hormone. Titration of human prolactin with the extracellular domain of the human prolactin receptor was followed by NMR, gel filtration and electrophoresis. Both binary and ternary hormone-receptor complexes are clearly detectable by gel filtration and electrophoresis. The binary complex is not observable by NMR, possibly due to a dynamic equilibrium in intermediate exchange within the complex. The ternary complex of one hormone molecule bound to two receptor molecules is on the contrary readily detectable by NMR. This is in stark contrast to the widely held view that the ternary prolactin-receptor complex is only transiently formed. Thus, our results lead to improved understanding of the prolactin-prolactin receptor interaction.

Amino Acid Sequence↗

PubMed2005

Enhancement of steroid receptor-mediated transcription for the development of highly responsive bioassays.

We have previously generated several transformed human mammary cell lines for the detection of steroid receptor-mediated activities and used these cell lines to detect and characterize steroid hormone (ant)agonistic compounds. In this report, we describe the specific optimization procedures used to enhance receptor-mediated transcription through the human glucocorticoid, progesterone and androgen receptors, respectively. Sodium arsenite-induced chemical stress leads to a substantial and specific increase in the glucocorticoid receptor-mediated transcription, resulting in maximal stimulations of more than 2000-fold by the agonist dexamethasone. Similarly, a combined treatment with forskolin (an activator of adenylate cyclase) and trichostatin A (an inhibitor of histone deacetylases) leads to a synergistic enhancement of progesterone or androgen stimulation, resulting in a maximal induction of more than 200-fold or about 100-fold, respectively. The enhanced responses to specific steroids are mediated by the corresponding nuclear receptor. We show that by using these enhanced transcriptional stimulation protocols, it is possible to detect lower amounts of steroid hormones without substantially affecting the relative biological activities of various agonists. Finally, the application of these enhanced reporter cell assays to real biological samples from meat-producing animals is evaluated, and some validation parameters are presented.

Animals↗

PubMed2005

The antiangiogenic factor, 16-kDa human prolactin, induces endothelial cell cycle arrest by acting at both the G0-G1 and the G2-M phases.

The 16-kDa N-terminal fragment of human prolactin (16K hPRL) is a potent antiangiogenic factor that has been shown to prevent tumor growth in a xenograph mouse model. In this paper we first demonstrate that 16K hPRL inhibits serum-induced DNA synthesis in adult bovine aortic endothelial cells. This inhibition is associated with cell cycle arrest at both the G(0)-G(1) and the G(2)-M phase. Western blot analysis revealed that 16K hPRL strongly decreases levels of cyclin D1 and cyclin B1, but not cyclin E. The effect on cyclin D1 is at least partially transcriptional, because treatment with 16K hPRL both reduces the cyclin D1 mRNA level and down-regulates cyclin D1 promoter activity. This regulation may be due to inhibition of the MAPK pathway, but it is independent of the glycogen synthase kinase-3beta pathway. Lastly, 16K hPRL induces the expression of negative cell cycle regulators, the cyclin-dependent kinase inhibitors p21(cip1) and p27(kip1). In summary, 16K hPRL inhibits serum-induced proliferation of endothelial cells through combined effects on positive and negative regulators of cell cycle progression.

Angiogenesis Inhibitors↗

PubMed2005

EGF stimulates Pit-1 independent transcription of the human prolactin pituitary promoter in human breast cancer SK-BR-3 cells through its proximal AP-1 response element.

Normal and neoplastic human mammary gland cells are targets for the proliferative action of prolactin. These cells also synthesize prolactin, thereby inducing an autocrine/paracrine proliferative loop. We present the first extensive analysis of the transcriptional regulation of the human prolactin gene (hPRL) in human mammary tumor cells, SK-BR-3. We show that the pituitary promoter is functional in these cells in the absence of the pituitary-specific factor Pit-1. Expression of exogenous Pit-1 or epidermal growth factor (EGF) treatment stimulates the transfected hPRL pituitary promoter and the endogenous hPRL expression. EGF stimulation is mediated by increased synthesis of c-fos and c-jun, resulting in AP-1 binding to the proximal hPRL pituitary promoter. This regulation involves the EGF receptor, possibly ErbB2 that is highly expressed in SK-BR-3 cells, and a PI3K/JNK pathway. The stimulation of hPRL gene transcription by EGF in mammary cells may include hPRL in a complex regulatory network controlling growth of human mammary cells.

Breast Neoplasms↗

PubMed2004

Cathepsin D processes human prolactin into multiple 16K-like N-terminal fragments: study of their antiangiogenic properties and physiological relevance.

16K prolactin (PRL) is the name given to the 16-kDa N-terminal fragment obtained by proteolysis of rat PRL by tissue extracts or cell lysates, in which cathepsin D was identified as the candidate protease. Based on its antiangiogenic activity, 16K PRL is potentially a physiological inhibitor of tumor growth. Full-length human PRL (hPRL) was reported to be resistant to cathepsin D, suggesting that antiangiogenic 16K PRL may be physiologically irrelevant in humans. In this study, we show that hPRL can be cleaved by cathepsin D or mammary cell extracts under the same conditions as described earlier for rat PRL, although with lower efficiency. In contrast to the rat hormone, hPRL proteolysis generates three 16K-like fragments, which were identified by N-terminal sequencing and mass spectrometry as corresponding to amino acids 1-132 (15 kDa), 1-147 (16.5 kDa), and 1-150 (17 kDa). Biochemical and mutagenetic studies showed that the species-specific digestion pattern is due to subtle differences in primary and tertiary structures of rat and human hormones. The antiangiogenic activity of N-terminal hPRL fragments was assessed by the inhibition of growth factor-induced thymidine uptake and MAPK activation in bovine umbilical endothelial cells. Finally, an N-terminal hPRL fragment comigrating with the proteolytic 17-kDa fragment was identified in human pituitary adenomas, suggesting that the physiological relevance of antiangiogenic N-terminal hPRL fragments needs to be reevaluated in humans.

Amino Acid Sequence↗

PubMed2004

Molecular targeting of antiangiogenic factor 16K hPRL inhibits oxygen-induced retinopathy in mice.

PURPOSE: To examine the ability and mechanism of the 16 kDa N-terminal fragment of human prolactin (16K hPRL) in the inhibition of abnormal retinal neovascularization. METHODS: The 16K hPRL-encoding sequence was inserted into an adenoviral vector (16K-Ad). Western blot analysis verified the expression of 16K hPRL and inhibition of proliferation, confirming functional activity of the 16K hPRL in virus-infected adult bovine aortic endothelial (ABAE) cells. 16K hPRL inhibited retinal neovascularization in a mouse model of oxygen-induced retinopathy. The ability of recombinant 16K hPRL expressed in E. coli (r16K hPRL) was compared to that of endostatin in inducing apoptosis of cultured human retinal endothelial cells (HREC). RESULTS: 16K was expressed in virus-infected ABAE cells and resulted in a dose-dependent inhibition of cell proliferation. Eyes injected with 16K-Ad showed a reduction in preretinal neovascularization of 82.3 +/- 9.3% (P < 0.00001) when compared to uninjected controls. r16K hPRL was 100 times more potent than endostatin in inducing apoptosis in HRECs. CONCLUSIONS: Intravitreal administration of 16K hPRL inhibited neovascularization in the mouse model of oxygen-induced retinopathy. 16K hPRL stimulated apoptosis in HRECs and inhibited cell proliferation in ABAE cells. These results suggested a potential therapeutic role for 16K hPRL in the treatment of proliferative retinopathies.

Adenoviridae↗

PubMed2003

Use of reporter cell lines for detection of endocrine-disrupter activity.

We have studied stable transformed human mammary cell lines with highly inducible steroid receptor-mediated luciferase reporter gene expression. Cells responding specifically to glucocorticoids, progestagens, androgens, or estrogens are described and characterized. The use of this high-throughput, cell-based assay for analysis of steroid (ant)agonists is reported. Systematic characterization of endocrine-disrupting activity on human receptors and in a human-cell system is interpreted for a selection of xenobiotics. We show that the phytoestrogens apigenin and genistin have progestagenic and androgenic activity, respectively. Finally, application of cell-based assays to the analysis of environmental samples is discussed.

Cell Line, Tumor↗

PubMed2003

Development of pure prolactin receptor antagonists.

Prolactin (PRL) promotes tumor growth in various experimental models and leads to prostate hyperplasia and mammary neoplasia in PRL transgenic mice. Increasing experimental evidence argues for the involvement of autocrine PRL in this process. PRL receptor antagonists have been developed to counteract these undesired proliferative actions of PRL. However, all forms of PRL receptor antagonists obtained to date exhibit partial agonism, preventing their therapeutic use as full antagonists. In the present study, we describe the development of new human PRL antagonists devoid of agonistic properties and therefore able to act as pure antagonists. This was demonstrated using several in vitro bioassays, including highly sensitive assays able to detect extremely low levels of receptor activation. These new compounds also act as pure antagonists in vivo, as assessed by analyzing their ability to competitively inhibit PRL-triggered signaling cascades in various target tissues (liver, mammary gland, and prostate). Finally, by using transgenic mice expressing PRL specifically in the prostate, which exhibit constitutively activated signaling cascades paralleling hyperplasia, we show that these new PRL analogs are able to completely revert PRL-activated events. These second generation human PRL antagonists are good candidates to be used as inhibitors of growth-promoting actions of PRL.

Amino Acid Motifs↗

PubMed2003

The antiangiogenic factor 16K human prolactin induces caspase-dependent apoptosis by a mechanism that requires activation of nuclear factor-kappaB.

We have previously shown that the 16-kDa N-terminal fragment of human prolactin (16K hPRL) has antiangiogenic properties, including the ability to induce apoptosis in vascular endothelial cells. Here, we examined whether the nuclear factor-kappaB (NF-kappaB) signaling pathway was involved in mediating the apoptotic action of 16K hPRL in bovine adrenal cortex capillary endothelial cells. In a dose-dependent manner, treatment with 16K hPRL induced inhibitor kappaB-alpha degradation permitting translocation of NF-kappaB to the nucleus and reporter gene activation. Inhibition of NF-kappaB activation by overexpression of a nondegradable inhibitor kappaB-alpha mutant or treatment with NF-kappaB inhibitors blocked 16K hPRL-induced apoptosis. Treatment with 16K hPRL activated the initiator caspases-8 and -9 and the effector caspase-3, all of which were essential for stimulation of DNA fragmentation. This activation of the caspase cascade by 16K hPRL was also NF-kappaB dependent. These findings support the conclusion that NF-kappaB signaling plays a central role in 16K hPRL-induced apoptosis in vascular endothelial cells.

Apoptosis↗

PubMed2002

Differential expression of two somatostatin genes during zebrafish embryonic development.

We have identified the cDNAs of two new zebrafish preprosomatostatins, PPSS1 and PPSS3, in addition to the previously cloned PPSS2 (Argenton et al., 1999). PPSS1 is the orthologue of mammalian PPSSs, with a conserved C-terminal SS-14 sequence, PPSS2 is a divergent SS precursor and PPSS3 is a cortistatin-like prohormone. Using whole-mount in situ hybridisation, we have analysed the expression of PPSS1 and PPSS2 in zebrafish embryos up to 5 days post fertilisation. PPSS1 was expressed in the developing pancreas and central nervous system (CNS), whereas PPSS2 expression was exclusively pancreatic. In the CNS, PPSS1 was detected in several areas, in particular in the vagal motor nucleus and in cells that pioneer the tract of the postoptic commissure. PPSS1 was also expressed transiently in the telencephalon and spinal motor neurons. In all areas but the telencephalon PPSS1 was coexpressed with islet-1.

Amino Acid Sequence↗

PubMed2002

Expression of the zinc finger Egr1 gene during zebrafish embryonic development.

Egr1 is a highly conserved zinc finger protein which plays important roles in many aspects of vertebrate development and in the adult. The cDNA coding for zebrafish Egr1 was obtained and its expression pattern was examined during zebrafish embryogenesis using whole-mount in situ hybridization. Egr1 mRNA is first detected in adaxial cells in the presomitic mesoderm between 11 and 20 h post-fertilization (hpf), spanning the 4-24 somite stages. Later, Egr1 expression is observed only in specific brain areas, starting at 21 hpf and subsequently increasing in distinct domains of the central nervous system, e.g. in the telencephalon, diencephalon and hypothalamus. Between 24 and 48 hpf, Egr1 is expressed in specific domains of the hypothalamus, mesencephalon, tegmentum, pharynx, retina, otic vesicle and heart.

Animals↗