Biomedical subjects

J F Martini

Publications and source records attributed to J F Martini.

14 recordsLinked to original sources

PubMed2001

Expression of the antiangiogenic factor 16K hPRL in human HCT116 colon cancer cells inhibits tumor growth in Rag1(-/-) mice.

The M(r) 16,000 NH(2)-terminal fragment of human prolactin (16K hPRL) is a potent antiangiogenic factor inhibiting endothelial cell function in vitro and neovascularization in vivo. The present study was undertaken to test the ability of 16K hPRL to inhibit the growth of human HCT116 colon cancer cells transplanted s.c. into Rag1(-/-) mice. For this purpose, HCT116 cells were stably transfected with an expression vector encoding a peptide that included the signal peptide and first 139 amino acid residues of human prolactin (HCT116(16K)). Stable clones of HCT116(16K) cells secreted large amounts of biologically active 16K hPRL into the culture medium. Growth of HCT116(16K) cells in vitro was not different from wild-type HCT116 (HCT116(wt)) or vector-transfected HCT116 (HCT116(vector)) cells. Addition of recombinant 16K hPRL had no effect on the proliferation of HCT116(wt) cells in vitro. Tumor growth of HCT116(16K) cells implanted into Rag1(-/-) mice was inhibited 63% in four separate experiments compared with tumors formed from HCT116(wt) or HCT116(vector) cells. Inhibition of tumor growth of HCT116(16K) cells was correlated with a decrease in microvascular density by 44%. These data demonstrate that biologically active 16K hPRL can be expressed and secreted from human colon cancer cells using a gene transfer approach and that production of 16K hPRL by these cells was capable of inhibiting tumor growth and neovascularization. These findings support the potential of 16K hPRL as a therapeutic agent for the treatment of colorectal cancer.

Animals↗

PubMed2001

IL-1beta induction of RANTES (regulated upon activation, normal T cell expressed and secreted) chemokine gene expression in endometriotic stromal cells depends on a nuclear factor-kappaB site in the proximal promoter.

A complex network of cytokines mediates immunoregulatory responses in the pathogenesis of endometriosis. RANTES (regulated upon activation, normal T cell expressed and secreted) is a chemoattractant for monocytes and T cells. Endometriotic lesions express RANTES, and its concentration in peritoneal fluid correlates with the severity of endometriosis. We investigated the influence of IL-1beta, a potent macrophage cytokine, on RANTES production in endometriotic stromal cells and determined the region of the RANTES promoter responsible for IL-1beta action. RANTES mRNA was induced 5-fold in endometriotic stromal cells, and the conditioned medium RANTES protein concentrations were 12-fold higher in IL-1beta-treated endometriotic stromal cells vs. untreated controls (P < 0.05). IL-1beta activated the full-length (-940 bp) RANTES promoter as well as a truncated 456-bp 5'-flanking construct by 2-fold. Mutagenesis of a nuclear factor-kappaB response element at -30 bp abolished the IL-1beta effect, whereas mutation of a nearby TNF response element did not affect the IL-1beta induction. An IL-1beta time-course Western assay revealed a rapid diminution of IkappaB (endogenous inhibitor of nuclear factor-kappaB) in endometriotic stromal cells. Overexpression of IkappaB in endometriotic stromal cells inhibited the IL-1beta response of the RANTES gene promoter. Transcription of RANTES mRNA is up-regulated by IL-1beta via a nuclear factor-kappaB response element in the proximal RANTES gene promoter. These results demonstrate a feed-forward regulatory loop in the pathogenesis of endometriosis by which IL-1beta produced from activated macrophages can lead to further macrophage recruitment via RANTES production in endometriotic stromal cells.

Blotting, Western↗

PubMed2000

Role of the cAMP signaling pathway in the regulation of gonadotropin-releasing hormone secretion in GT1 cells.

We studied the signaling pathways coupling gonadotropin-releasing hormone (GnRH) secretion to elevations in cAMP levels in the GT1 GnRH-secreting neuronal cell line. We hypothesized that increased cAMP could be acting directly by means of cyclic nucleotide-gated (CNG) cation channels or indirectly by means of activation of cAMP-dependent protein kinase (PKA). We showed that GT1 cells express the three CNG subunits present in olfactory neurons (CNG2, -4.3, and -5) and exhibit functional cAMP-gated cation channels. Activation of PKA does not appear to be necessary for the stimulation of GnRH release by increased levels of cAMP. In fact, pharmacological inhibition of PKA activity caused an increase in the basal secretion of GnRH. Consistent with this observation activation PKA inhibited adenylyl cyclase activity, presumably by inhibiting adenylyl cyclase V expressed in the cells. Therefore, the stimulation of GnRH release by elevations in cAMP appears to be the result of depolarization of the neurons initiated by increased cation conductance by cAMP-gated cation channels. Activation of PKA may constitute a negative-feedback mechanisms for lowering cAMP levels. We hypothesize that these mechanisms could result in oscillations in cAMP levels, providing a biochemical basis for timing the pulsatile release of GnRH.

Adenylyl Cyclases↗

PubMed2000

The antiangiogenic factor 16K PRL induces programmed cell death in endothelial cells by caspase activation.

We asked whether the antiangiogenic action of 16K human PRL (hPRL), in addition to blocking mitogen-induced vascular endothelial cell proliferation, involved activation of programmed cell death. Treatment with recombinant 16K hPRL increased DNA fragmentation in cultured bovine brain capillary endothelial (BBE) and human umbilical vein endothelial (HUVE) cells in a time- and dose-dependent fashion, independent of the serum concentration. The activation of apoptosis by 16K hPRL was specific for endothelial cells, and the activity of the peptide could be inhibited by heat denaturation, trypsin digestion, and immunoneutralization, but not by treatment with the endotoxin blocker, polymyxin-B. 16K hPRL-induced apoptosis was correlated with the rapid activation of caspases 1 and 3 and was blocked by pharmacological inhibition of caspase activity. Caspase activation was followed by inactivation of two caspase substrates, poly(ADP-ribose) polymerase (PARP) and the inhibitor of caspase-activated deoxyribonuclease (DNase) (ICAD). Furthermore, 16K hPRL increased the conversion of Bcl-X to its proapoptotic form, suggesting that the Bcl-2 protein family may also be involved in 16K hPRL-induced apoptosis. These findings support the hypothesis that the antiangiogenic action of 16K hPRL includes the activation of programmed cell death of vascular endothelial cells.

Antibodies, Monoclonal↗

PubMed1999

Ischemic preconditioning attenuates ischemia/reperfusion-induced activation of caspases and subsequent cleavage of poly(ADP-ribose) polymerase in rat hearts in vivo.

Recently, we have demonstrated that ischemic preconditioning (IP) both limits infarct size and decreases internucleosomal DNA fragmentation in rat hearts in vivo, and that there was a direct correlation between myocardial infarct size and DNA fragmentation even after IP. In this study, we examined the ability of IP to attenuate processing and activation of caspase-1 and caspase-3, and cleavage of poly(ADP-ribose) polymerase (PARP), after prolonged ischemia and reperfusion using the same in vivo animal model. Rats that underwent IP and controls (Ctrl) were subjected to 30 min of left coronary artery occlusion followed by 180 min of reperfusion. IP was accomplished by five 5-min cycles of ischemia, each followed by 5 min of reperfusion. The amount of soluble nucleosomes was measured by enzyme-linked immunosorbent assay. Cleavage of caspases-1 and -3, and of one of their substrates PARP, was analyzed by Western blotting. Nucleosomal DNA fragmentation was significantly reduced in ischemic left ventricular (LV) tissue obtained from IP compared with Ctrl animals. The proforms of caspases-1 and -3, and the active form of PARP were not cleaved in the nonischemic LV region of both IP and Ctrl hearts. In contrast, the proform of caspase-3 and the active form of PARP were cleaved in the ischemic LV region of Ctrl hearts, while processing of caspase-1 was increased. Cleavages of caspases-1 and -3, and inactivation of PARP were prevented by IP. The results of this study indicate that IP attenuates both internucleosomal DNA fragmentation and caspases processing, and suggest that the prevention of caspases activation by IP may be important steps in protecting the heart against ischemia/reperfusion injury in vivo.

Analysis of Variance↗

PubMed1999

16K human prolactin inhibits vascular endothelial growth factor-induced activation of Ras in capillary endothelial cells.

Signaling pathways mediating the antiangiogenic action of 16K human (h)PRL include inhibition of vascular endothelial growth factor (VEGF)-induced activation of the mitogen-activated protein kinases (MAPK). To determine at which step 16K hPRL acts to inhibit VEGF-induced MAPK activation, we assessed more proximal events in the signaling cascade. 16K hPRL treatment blocked VEGF-induced Raf-1 activation as well as its translocation to the plasma membrane. 16K hPRL indirectly increased cAMP levels; however, the blockade of Raf-1 activation was not dependent on the stimulation of cAMP-dependent protein kinase (PKA), but rather on the inhibition of the GTP-bound Ras. The VEGF-induced tyrosine phosphorylation of the VEGF receptor, Flk-1, and its association with the Shc/Grb2/Ras-GAP (guanosine triphosphatase-activating protein) complex were unaffected by 16K hPRL treatment. In contrast, 16K hPRL prevented the VEGF-induced phosphorylation and dissociation of Sos from Grb2 at 5 min, consistent with inhibition by 16K hPRL of the MEK/MAPK feedback on Sos. The inhibition of Ras activation was paralleled by the increased phosphorylation of 120 kDa proteins comigrating with Ras-GAP. Taken together, these findings show that 16K hPRL inhibits the VEGF-induced Ras activation; this antagonism represents a novel and potentially important mechanism for the control of angiogenesis.

Biological Transport↗

PubMed1998

Increased expression of prolactin receptor gene assessed by quantitative polymerase chain reaction in human breast tumors versus normal breast tissues.

The role of PRL in human breast tumorigenesis is not well understood. One of the limitations is the difficulty of accurately measuring PRL receptors (PRLR) in human tissues. We established a quantitative PCR method (Q-PCR) in T-47D human breast cancer cells and applied it to 29 patients, 25 of whom presented with either cancer or fibroadenoma. Four patients underwent a mammoplasty, and normal epithelial cells were cultured before Q-PCR. In T-47D cells, 31 x 10(6) messenger RNA molecules were detected per microgram of total RNA. In all patients, expression of the PRLR gene was detected, varying from 1500 to 1 x 10(6) molecules/microgram of RNA in normal tissues and from 4500 to 34.7 x 10(6) molecules/microgram of RNA in tumors. PRLR expression was always greater in tumor than in normal contiguous tissue and similar in cultured mammary epithelial cells and normal breast tissues. Estradiol and progesterone receptor-negative tumors expressed low levels of PRLR transcripts, similar to normal breast tissue from menopausal women. Immunocytochemical analysis of PRLR confirmed stronger staining in almost all tumor samples compared with normal tissues. A messenger RNA encoding locally produced human PRL was also identified by RT-PCR in every sample tested. Our results confirm PRLR gene expression in all tissues studied, and moreover, indicate that this expression is increased in human breast tumors vs. normal contiguous tissues.

Adult↗

PubMed1998

The D152H mutation found in growth hormone insensitivity syndrome impairs expression and function of human growth hormone receptor but is silent in rat receptor.

In two patients with growth hormone (GH) insensitivity syndrome (Laron syndrome), in whom the GH receptor is able to bind the hormone, the D152H mutation was identified, and lack of dimerization was proposed to explain GH resistance in these patients. To examine further the consequences of the substitution of conserved aspartate 152 on the function of the GH receptor (GHR), we reproduced the mutation in vitro on the full length GH receptor cDNA from man and rat. Effects of the mutation on expression and activity of the GHR were analyzed in 293 cells transfected with wild-type and mutant GHR cDNAs. Mutant human receptor protein was expressed at a lower level than wild-type receptor and its activity was reduced: GH-dependent signal transducer and activator of transcription 5 (Stat5)-mediated transactivation of a reporter gene was lower in 293 cells transfected with mutant GHR cDNA than in transfected cells expressing a comparable level of wild-type GHR. The membrane-bound form of the mutant and of the wild-type human GHR were able to homodimerize, as suggested by the size of the complexes detected in cross-linking experiments with 125I-human (h) GH, and also by the activity in the functional test. With the soluble GHR resulting from proteolysis of the wild-type membrane form, no dimeric complexes could be detected. However, when a soluble receptor lacking the transmembrane and cytoplasmic domains of the receptor was expressed, wild-type and not mutant GH binding protein (GHBP) was able to form dimers in the presence of hGH. The amino acid substitution has no effect on either expression or function of the rat receptor. Structural modeling of D152H soluble human and rat GHR (GHBP) supports the species-specific functional consequences of the mutation. Evaluation of the functional importance of the mutation strongly suggests that impairment in expression and activity of the mutant receptor, rather than complete lack of dimerization, explains the GH resistance of the patients.

Animals↗

PubMed1997

Monkey growth hormone (GH) receptor gene expression. Evidence for two mechanisms for the generation of the GH binding protein.

The growth hormone receptor (GHR) cDNA was cloned from the liver of Rhesus macaque using polymerase chain reaction. As deduced from the nucleotide sequence, the mature GHR is a protein of 620 amino acids which presents 94.1% identity with the human receptor. The monkey GHR (mkGHR) expressed in 293 cells presented the expected specificity for a primate GHR and was able to transduce a transcriptional effect of GH. Human GH was able to activate tyrosine phosphorylation of both the tyrosine kinase JAK2 and the receptor in 293 cells co-transfected with mkGHR and JAK2 cDNAs. The GH binding protein (GHBP), the soluble short form of the GHR, was also present in monkey serum. Expression of the GHR cDNA in eucaryotic cells indicated that the GHBP can be produced by proteolytic cleavage of the membrane receptor. Northern blot analysis of GHR gene expression in different tissues allowed us to identify three different transcripts of 5.0 and 2.8 kilobase pairs and a smaller one of 1.7 kilobase pairs which could encode a GHBP. Rapid amplification of cDNA extremities (3'-RACE-polymerase chain reaction) was used to identify a cDNA encoding a protein in which the transmembrane and cytoplasmic domains of the receptor are substituted by a short sequence of 9 amino acids. This transcript was present in various tissues and could encode a GHBP as well, suggesting for the first time that two different mechanisms can coexist for the generation of the GHBP: proteolytic cleavage of the membrane receptor and a specific mRNA produced by alternative splicing.

Alternative Splicing↗

PubMed1995

Quantitative analysis by polymerase chain reaction of growth hormone receptor gene expression in human liver and muscle.

A single form of GH receptor (GHR) messenger RNA (mRNA) of 4.5 kilobases, encoding the full-length GHR, has been found in man. To measure the absolute number of mRNA molecules encoding the GHR in human tissues, we developed a quantitative polymerase chain reaction assay. An internal control RNA was constructed by inserting a 50-basepair fragment of the rat PRL receptor complementary DNA into a portion of the human GHR complementary DNA. The internal control RNA and the target mRNA were amplified together with the same set of primers. Twenty-four cycles of amplification were used to satisfy an exponential phase of amplification. It was possible to detect as few as 500 molecules of target mRNA/micrograms total RNA. In 3 liver samples obtained from normal donors at the time of transplant, the amount of GHR mRNA ranged from 0.5 +/- 0.1 to 1.4 +/- 0.4 x 10(6) molecules/micrograms total RNA. These results were confirmed by slot blot analysis of the same samples. The number of receptor transcripts did not appear to be correlated with the receptor-binding capacity found in the 3 liver samples. In 7 muscle biopsies, GH receptor mRNA varied between 4.0 +/- 0.4 and 34.6 +/- 1.4 x 10(4) molecules/micrograms total RNA. This technique allows direct measurement of GHR gene expression in human tissues and represents a valuable tool, particularly for tissues such as muscle, in which the receptor protein cannot be measured using conventional binding assays.

Base Sequence↗

PubMed1995

[Growth hormone receptor. Structure and signal transduction].

The growth hormone receptor (GHR) belongs to the family of the prolactin and cytokine receptors. The full length receptor in a 620 amino acid protein with a unique transmembrane domain. The GH binding protein (GHBP) corresponds to the extracellular domain of the membrane GHR. In all human tissues tested, one form of 4.5 kb for the GHR mRNA was detected, suggesting that GHBP is generated through proteolytic cleavage of the membrane receptor. The three dimensional crystollographic structure of GHBP-hGH complex has identified a homodimer made of two receptor molecules and one molecule of hGH. Hormone-induced receptor dimerisation appears to be crucial for signal transduction. Functional tests using the GH effect on transcription of genes, such as SP12.1 and beta lactoglobulin, have been developed to define the sequences of the receptor which are important for signaling. A proline-rich juxtamembranous sequence, called Box 1, is important for GH effects on gene transcription, on MAP kinase activity, on cell proliferation, and on JAK2 activation. JAK2 has been identified to be a GHR-associated tyrosine kinase. The first 46 amino acids of the cytoplasmic domain are necessary for JAK2 and MAP kinase activation whereas a C-Ter sequence is necessary for the transcriptional effect. Substrates for JAK2, other than the receptor itself, have to be identified. Good candidates are the transcription factors STAT.

Humans↗

PubMed1994

Reduced food intake is the main cause of low growth hormone receptor expression in uremic rats.

To examine the respective effects of reduced food intake and of uremia on the growth defect in uremic rats, we have studied the expression of GH receptors in three groups of male rats: Group 1, rats fed ad libitum; Group 2, food-restricted to be pair-fed with uremic rats; Group 3, uremic rats. Animals were studied for a time period of 9 days starting 1 week after surgery (sham operation in rats of Groups 1 and 2, 5/6 nephrectomy in rats of Group 3). The gain in body length and weight of pair-fed controls and of uremic rats was comparable and significantly lower than that of rats fed ad libitum. IGF-1 plasma levels were low in rats of groups 2 and 3. Low food intake (50% that of rats fed ad libitum) resulted in a reduced number of GH receptors in liver membranes and a low plasma level of GH-binding protein (GHBP); GH receptor gene expression in the liver, as analyzed by Northern blots, was not significantly lower in normal food-restricted animals. In uremic rats, the low level of GH binding to liver membranes was comparable to that found in pair-fed controls; but the level of GHBP activity was normal, not different from the values found in rats fed ad libitum. However, expression of the liver GHBP mRNA was reduced in uremic rats. In uremia, the GH receptor dysfunction is not only at a transcriptional level but also at a post-transcriptional level. These findings suggest that uremia, as such, is not primarily responsible for the growth failure.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Expression of the growth hormone receptor gene in human digestive tissue.

We have examined the forms and the distribution of the messenger ribonucleic acids (mRNAs) encoding the GH receptor (GHR) in human digestive tissues. GHR mRNAs were identified and characterized by Northern blot, dot blot, and in situ hybridization analyses, using complementary DNAs coding for the extracellular part of the human liver GHR. Amplification using the polymerase chain reaction was also used, as tissues express low levels of GHR mRNAs. Our results demonstrate that the GHR gene is expressed in human liver, pancreas, esophagus, stomach, small intestine, and colon. A single 4.5-kilobase mRNA form, which probably encodes the full-length membrane receptor, was detected. GHR mRNA was visualized by in situ hybridization in hepatocytes, exocrine pancreas, and some islet cells; signal was also present in the mucosa of the digestive tract. No specific GH-binding-protein mRNA was found, suggesting that in man, the soluble form of the receptor is generated through proteolytic cleavage of the membrane receptor.

Adolescent↗

PubMed1991

[Growth hormone receptor].

Growth hormone (GH) exerts multiple actions and GH receptors have been demonstrated in a variety of tissues. Changes in the number of hepatic GH receptors have been demonstrated in several models of growth failure in rats. Cloning of the rabbit and human liver GH receptor has shown that the receptor is a single polypeptide chain of 620 amino acids, made of an extracellular hormone-binding domain, with 7 cysteines and 5 potential glycosylation sites, a unique transmembrane domain and a long cytoplasmic domain. The GH receptor belongs to a new family including prolactin and cytokine receptors. Signal transduction pathways are unknown for these receptors, which do not possess any consensus sequences homologous to tyrosine kinases. The GH-binding protein (GH-BP), identified in plasma of man and other species, corresponds to the extracellular binding domain of the membrane GH receptor. Evaluation of the GH-BP is a direct approach to the GH receptor in man in vivo. Complete absence of GH binding activity has been found in the plasma of patients with Laron dwarfism, in particular those for whom a mutation in the GH receptor gene has been demonstrated.

Animals↗