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Biomedical subjects

J J Feige

Publications and source records attributed to J J Feige.

At least 19 recordsLinked to original sources
PubMed2001

Thrombospondins and tumor angiogenesis.

The thrombospondins (TSPs) are a family of five secreted proteins that are widely distributed in the extracellular matrix of numerous tissues. TSPs are multimodular and each domain specifies a distinct biological function through interaction with a specific receptor. TSP1 and TSP2 have anti-angiogenic activity, which, at least for TSP1, involves interaction with the microvascular endothelial cell receptor CD36. Expression of TSP1 and TSP2 is modulated by hypoxia and by oncogenes. In several tumors (thyroid, colon, bladder carcinomas), TSP1 expression is inversely correlated with tumor grade and survival rate, whereas in others (e.g. breast carcinomas), it is correlated with the stromal response and is of little prognostic value. Recent studies suggest that TSPs or TSP-derived peptides retaining biological activity could be developed into promising new therapeutic strategies for the anti-angiogenic treatment of solid tumors.

Animals↗
PubMed2000

Thrombospondin-1 is downregulated by anoxia and suppresses tumorigenicity of human glioblastoma cells.

Angiogenesis, the sprouting of new capillaries from preexisting blood vessels, results from a disruption of the balance between stimulatory and inhibitory factors. Here, we show that anoxia reduces expression of thrombospondin-1 (TSP-1), a natural inhibitor of angiogenesis, in glioblastoma cells. This suggests that reduced oxygen tension can promote angiogenesis not only by stimulating the production of inducers, such as vascular endothelial growth factor, but also by reducing the production of inhibitors. This downregulation may significantly contribute to glioblastoma development, since we show that an increase in TSP-1 expression is sufficient to strongly suppress glioblastoma cell tumorigenicity in vivo.

Animals↗
PubMed2000

Expression and regulation of melanocortin receptor-5 (MC5-R) in the bovine adrenal cortex.

Among the five members of the melanocortin receptor (MC-R) family, MC2 and MC5 are expressed in peripheral tissues. The receptor MC2 (ACTH receptor) almost exclusively expressed in the adrenal cortex whereas MC5-R is expressed in several organs including the adrenal cortex. Both receptors bind ACTH and activate adenylate cyclase. The aim of this work was to study the spatial distribution of MC5-R among the different zones of the bovine adrenal cortex and to analyze the regulation of its expression by its own ligands, ACTH and alpha-MSH and by angiotensin II (AII). Using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and RNase protection assay, MC5-R was detected only in the glomerulosa zone whereas MC2-R was present in both glomerulosa and fasciculata zones of adult adrenal cortex. Treatments by ACTH, alpha-MSH, or AII increased the MC5-R mRNA level in glomerulosa cells by factors 7, 5, and 4.5, respectively. However, although potentially regulated by hormones, MC5-R is expressed at a level at least 100 times less than MC2-R, suggesting that MC5-R expression might only be at trace levels in grown adults, but could be much higher during embryogenesis.

Adrenal Cortex↗
PubMed2000

ACTH-regulated expression of vascular endothelial growth factor in the adult bovine adrenal cortex: a possible role in the maintenance of the microvasculature.

Endothelial cells lining vessels of endocrine tissues are fenestrated. Interactions with the local environment via either soluble factors or cell-cell interactions appear to govern this terminal endothelial differentiation. Adrenocorticotropin (ACTH) has previously been reported to modulate endothelial fenestration in the rat adrenal cortex. Since vascular endothelial growth factor (VEGF) has been characterized as a potent inducer of endothelial fenestration, we aimed to characterize the status of VEGF expression in the bovine adult adrenal cortex and asked whether ACTH may regulate VEGF expression. By immunohistochemical analysis, we observed VEGF expression in steroidogenic cells from both zona glomerulosa and zona fasciculata of the bovine adrenal cortex. Double-labeling experiments performed on isolated cells in primary culture revealed VEGF immunoreactivity, essentially colocalized with the Golgi apparatus. The expression of two predominant VEGF isoforms, VEGF(121) and VEGF(165), was observed by RT-PCR analysis. ACTH (10 nM) was found to rapidly (within 2-4 h) increase the abundance of these VEGF transcripts, as assessed by both RT-PCR and Northern blot analysis. In parallel, ACTH significantly induced VEGF secretion into the medium of fasciculata cells in primary culture. Thus, our data are consistent with the involvement of ACTH, through its regulation of VEGF expression, in the maintenance of the adult adrenal cortex endothelium.

Adrenal Cortex↗
PubMed2000

Transforming growth factor-beta receptor types I and II in cultured porcine leydig cells: expression and hormonal regulation.

The steroidogenic activity of testicular Leydig cells is controlled both by the pituitary hormone (LH) and by growth factors such as transforming growth factor-beta peptides (TGFbeta1, -2, and -3; inhibin/activin; and anti-Mullerian hormone). By using primary cultures of porcine Leydig cells as a model, the aim of the study was to identify and characterize the TGFbeta receptors and to study their regulation by LH/hCG. TGFbeta receptors have been identified and characterized through three different approaches, including cross-linking experiments and Western and Northern blotting analyses. In cross-linking experiments, labeled TGFbeta was shown to bind to three different molecular species of 300, 80, and 53 kDa, which may correspond to the protein betaglycan (also known as TGFbeta type III receptor) and TGFbeta type II and I receptors (TGFbetaRII and TGFbetaRI), respectively. The presence of TGFbetaRI and -RII was further demonstrated by Western blotting analysis using specific polyclonal antibodies. Finally, the expression of betaglycan, TGFbetaRII, and TGFbetaRI messenger RNAs, was confirmed by Northern blotting analysis, as shown by the presence of 6.4-, 4.6-, and 5.8-kb messenger RNAs, respectively. By using a RT-PCR approach, the mediators of the TGFbeta signal, Smads 1-7, were also detected in cultured Leydig cells. TGFbetaRI and TGFbetaRII protein levels were enhanced by hCG/LH in a dose-dependent (maximal effect with 0.3 ng/ml hCG) and time-dependent (maximal effect observed after 48 h of hCG treatment) manner. Furthermore, to determine whether the stimulatory effect of LH/hCG was mediated by testosterone, use was made of aminogluthetimide, an inhibitor of cytochrome P450scc. The inhibition oftestosterone formation did not affect the stimulatory effect of LH/hCG on TGFbetaRI and -RII levels, suggesting that the gonadotropin action is not mediated by the steroid hormone. Together, the present findings demonstrate that the TGFbeta receptors are expressed and are under hormonal (gonadotropin) control in cultured porcine Leydig cells.

Aminoglutethimide↗
PubMed2000

Fibroblast growth factor-2 inhibits the maturation of pro-insulin-like growth factor-II (Pro-IGF-II) and the expression of insulin-like growth factor binding protein-2 (IGFBP-2) in the human adrenocortical tumor cell line NCI-H295R.

The IGF system is thought to play a major role in adrenocortical tumorigenesis. In this study, we used the NCI H295R cell line as a model to investigate the effects of fibroblast growth factor-2 (FGF-2), a potent mitogen for normal adrenal cells, on the proliferation and on the expression of the IGF system in cultured adrenocortical tumor cells. Three immunoreactive FGF-2 isoforms of molecular masses 18, 22, and 24 kDa were detected in H295R cell extracts. Recombinant human FGF-2 stimulated the proliferation of adrenocortical tumor cells in a dose- and time-dependent manner, with a maximal effect at a concentration of about 1 ng/ml. Treatment of H295R cells with 10 ng/ml FGF-2 for 7 days had no significant effect on IGF-II messenger RNA levels. However, a marked increase in levels of intracellular IGF-II protein was detected by immunoblotting. In contrast, FGF-2 induced a marked decrease in the amount of IGF-II protein secreted, with the disappearance of mature IGF-II and secretion of higher molecular forms of the growth factor, suggesting modifications of IGF-II processing. Cell cultures in the presence of brefeldin A (1 microg/ml), a specific inhibitor of protein secretion, suggested that FGF-2 did not increase IGF-II synthesis but instead inhibited the secretion of pro-IGF-II from H295R cells, thereby impairing the final steps of IGF-II processing to the mature 7.5-kDa peptide. At the same concentrations, FGF-2 also decreased both IGFBP-2 messenger RNA and secreted protein, which might increase IGF-II bioavailability. No proteolysis of IGFBP-2 was detected in FGF-2-conditioned medium. Altogether, these results indicate that FGF-2 is mitogenic for NCI H295R tumor cells and regulates the expression of both IGF-II and IGFBP-2 in this tumor model. Moreover, this study shows a novel effect of FGF-2, by which this growth factor modulates the processing of pro-IGF-II.

Adrenal Cortex Neoplasms↗
PubMed2000

Expression of the angiogenesis markers vascular endothelial growth factor-A, thrombospondin-1, and platelet-derived endothelial cell growth factor in human sporadic adrenocortical tumors: correlation with genotypic alterations.

Several studies have supported the hypothesis that adrenocortical tumor formation is the result of a multistep process. The angiogenic switch has been proposed to be a key step in tumor progression from adenoma to carcinoma. In this study we measured the cytosolic concentrations of three proteins involved in angiogenesis [namely platelet-derived endothelial cell growth factor vascular endothelial cell growth factor A (VEGF-A), and thrombospondin-1 (TSP1)] in a series of 43 human sporadic adrenocortical tumors. The tumors were classified as adenomas (n = 18), transitional tumors (n = 12), or carcinomas (n = 13) according to the histological criteria defined by Weiss. Platelet-derived endothelial cell growth factor/thymidine phosphorylase levels were not significantly different among these three groups. One hundred percent of the adenomas and 73% of the transitional tumors showed VEGF-A concentrations under the threshold value of 107 ng/g protein, whereas 75% of the carcinomas had VEGF-A concentrations above this threshold value. Similarly, 89% of the adenomas showed TSP1 concentrations above the threshold value of 57 microg/g protein, whereas only 25% of the carcinomas and 33% of the transitional tumor samples did so. Insulin-like growth factor II overexpression, a common genetic alteration of adrenocortical carcinomas, was significantly correlated with higher VEGF-A and lower TSP1 concentrations. The tumors from the 6 patients with tumor recurrence after surgical ablation showed significantly higher VEGF-A values than the carcinomas and the transitional tumors from patients that did not relapse. Taken together, these data suggest that a decrease in TSP1 expression is an event that precedes an increase in VEGF-A expression during adrenocortical tumor progression. The population of premalignant tumors with low TSP1 and normal VEGF-A levels could represent a selective target for antiangiogenic therapies.

Adrenal Cortex Neoplasms↗
PubMed2000

Paracrine control of the adult adrenal cortex vasculature by vascular endothelial growth factor.

The extensive vascular network that irrigates the adult adrenal cortex is essential for both the delivery of oxygen and nutrients to glandular steroidogenic cells and for the rapid and efficient export of corticosteroid products from these cells into the blood flow. During experimental and pathological changes in adrenal cortex size caused by ACTH overproduction or deficiency, the vasculature evolves in a coordinated manner with the mass of glandular cells so that blood vessel formation/regression and cortical gowth/atrophy are respectively synchronized. In addition to its previously reported expression in human fetal adrenocortical cells, the angiogenic factor VEGF-A appears also to be strongly expressed by both glomerulosa and fasciculata cells of the adult bovine adrenal cortex, when the endothelium is quiescent. Moreover, the expression of the two major transcripts encoding the 121 and the 165 amino acid-long isoforms of VEGF-A was observed to be rapidly (within 2-4 h) up-regulated (2-3 fold) by ACTH in primary cultures of bovine fasciculata and glomerulosa cells. The expression of the signalling VEGF receptors R1 (flt-1) and R2 (flk-1) was restricted to the endothelial cells of the cortex whereas neuropilin-1 was expressed by both endothelial and steroidogenic cells. This suggests that, under the control of the pituitary hormone ACTH, VEGF exerts a paracrine control over the vasculature of the adult adrenal cortex. Given its known effects as an anti-apoptotic agent and an inducer of endothelial fenestration, VEGF is likely to play a role in the maintenance of the dense and fenestrated vascular bed of the adrenal cortex. The vasculature thus appears as an important secondary target of ACTH action in the physiological control of adrenal cortex homeostasis.

Adrenal Cortex↗
PubMed2000

Transcriptional regulation of the gene encoding the StAR protein in the human adrenocortical cell line, H295R by cAMP and TGFbeta1.

In the present work, we have analyzed the transcriptional regulation of StAR expression in the human adrenocortical cell line H295R. We observed that StAR mRNA levels rapidly increased in response to forskolin, starting after 2 h of treatment and reaching a maximum by 12 h. Deletion analysis of the human StAR promoter demonstrated that the first 150 bp upstream of the transcription start were sufficient for both basal and cAMP-induced expression of a reporter gene. We demonstrate that out of the three binding sequences for the steroidogenic factor 1 (SF-1) only the central one (-105 to -96) is implicated in both basal and cAMP-induced StAR expression. In addition, another important regulatory element for both basal and cAMP-induced StAR expression is present between -62 and -24 upstream of the transcription start. We also show that TGF1beta1 inhibits StAR expression at the transcriptional level. We found that the TGFbeta-inhibitory element lies between -150 and -85 upstream of the transcription start and that the SF-1 binding sites are not implicated in TGFbeta1 regulation.

Adrenal Cortex↗
PubMed2000

[Molecular bases of angiogenesis].

Angiogenesis is a multistep process that drives the formation of new blood capillaries from the pre-existing vasculature. It is usual to distinguish two phases in this process: a morphogenic phase that requires endothelial cell proliferation and migration followed by a maturation phase characterized by the reconstitution of extracellular matrix and the recruitment of pericytes around the newly formed microvessel. Angiogenesis is actively involved in embryonic organogenesis as well as in cancer tumor growth. Although the properties of neovessels formed under these two situations are different, the molecules and the mechanisms implicated are common. VEGF and its receptors flk-1 and flt-1 are the main actors of the morphogenic phase whereas PDGF-BB and its receptor PDGF-R beta, TGF beta 1 and its receptors ALK-5 and ALK-1, angiopoietin-1 and its receptor tie-2 are implied in the vascular maturation phase.

Animals↗
PubMed1999

Tissue inhibitor of metalloproteinase-2 (TIMP-2) expression is strongly induced by ACTH in adrenocortical cells.

Besides its acute and chronic effects on corticosteroid synthesis, the pituitary adrenocorticotropic hormone (ACTH) regulates diverse adrenocortical biological functions including the synthesis of a number of mitochondrial, cytoplasmic, and secreted proteins. ACTH-induced secreted proteins are candidates to act as local extracellular relays of the hormone in either an autocrine or a paracrine manner. In the present study, we report that stimulation of primary cultures of bovine adrenocortical (BAC) fasciculata cells with 10 nM ACTH for 24 h results in a mean 8 +/- 4-fold induction of the synthesis of a secreted protein presenting an apparent Mr of 21 kDa. Peptide microsequencing and Western blotting allowed us to identify this 21-kDa ACTH-induced protein as the tissue inhibitor of metalloproteinase-2 (TIMP-2). The induction of TIMP-2 by ACTH required transcription, was mimicked by 8-bromo cyclic 3'-5' adenosine monophosphate, but was not observed in response to angiotensin II, IGF-1, fibroblast growth factor-2, transforming growth factor-beta1, or cortisol treatments. ACTH stimulated TIMP-2 mRNA levels by a factor 4, whereas TIMP-1 mRNA levels were not affected and TIMP-3 mRNA remained undetectable. The biological activity of TIMP-2 varied accordingly, as we observed that the conditioned medium of ACTH-treated BAC cells was four times more potent at inhibiting gelatinolytic activity than was the conditioned medium of control cells. Because the proteolytic activity of both progelatinase-B and progelatinase-A secreted by BAC cells remained latent, whether in the presence or in the absence of ACTH, a paracrine rather than autocrine role is proposed for TIMP-2 in the adrenal cortex.

Adrenal Cortex↗
PubMed1999

Regulation of the three-dimensional organization of thyroid epithelial cells into follicle structures by the matricellular protein, thrombospondin-1.

Thyroid epithelial cells in primary culture have the capacity to organize into thyroid-specific three-dimensional structures, the follicles, in response to TSH. We studied whether thrombospondin 1 (TSP1), which represents, besides thyroglobulin, the main protein secreted by thyroid cells, could play a role in the process of folliculogenesis. TSH promoted follicle formation and inhibited TSP1 production. On the contrary, the phorbol ester, 12-O-tetradecanoyl-phorbol 13-acetate (1-100 nM) prevented TSH-induced follicle formation and strongly increased the synthesis of TSP1. Activation of TSP1 synthesis was dependent upon messenger RNA synthesis. Transforming growth factor-beta, like 12-O-tetradecanoyl-phorbol 13-acetate, increased TSP1 synthesis and prevented TSH-induced follicle formation. Thus, signaling molecules that depressed or conversely activated TSP1 production, respectively promoted or prevented thyroid folliculogenesis. TSP1, purified from platelets, was devoid of effect on cell substratum attachment, but exerted a concentration-dependent inhibition of the TSH-activated reconstitution of thyroid follicles (half-inhibition at 40 microg/ml). TSP1 exhibited the same effect when added to thyroid cell aggregates representing primitive follicle structures. Our data suggest that the control of thyroid follicle formation may operate at least in part through regulation of the production of the matricellular protein TSP1, which acts as a negative modulator of the cell-cell adhesion process involved in thyroid follicle morphogenesis.

Animals↗
PubMed1999

Bovine thrombospondin-2: complete complementary deoxyribonucleic acid sequence and immunolocalization in the external zones of the adrenal cortex.

Given the variety of biological functions in the adrenal cortex that are controlled by ACTH, we hypothesized that some extracellular proteins act as biological relays for this systemic hormone. One candidate protein [corticotropin-induced secreted protein (CISP)] was purified from the conditioned medium of bovine adrenocortical cells on the basis of a 5- to 14-fold increase in its synthesis after the addition of ACTH. We report here the cloning of overlapping complementary DNAs that span the sequence encoding the full-length protein (1170 amino acids). The deduced CISP protein sequence is 89% identical to that of human thrombospondin-2 (TSP2), but only 61% identical to that of bovine TSP1, confirming that CISP is the bovine ortholog of TSP2. The bovine TSP2 sequence aligned perfectly with human, mouse, and chicken TSP2 sequences, except for a gap of 2 amino acids located in a linker region. All 58 cysteine residues that are conserved in other species were present in the bovine sequence as well as most of the functional domains. Most endocrine tissues (adrenal cortex, testis, ovary, and placenta) appeared to express TSP2, as determined by Western blot analysis. The highest levels of TSP2 protein were found in the adrenal cortex, followed by the heart, spleen, brain, and kidney. A differential extent of N-glycosylation or tissular proteolytic maturation may be responsible for the mol wt differences observed between bovine TSP2 detected in the medium from primary cultures and that in fresh tissue extracts. The immunohistochemical analysis of the distribution of TSP2 in the bovine adrenal gland revealed that the protein is much more abundant in the external zones (zona glomerulosa and zona fasciculata) than in the internal reticularis zone, a pattern similar to that reported for ACTH receptors. This distribution clearly suggests that TSP2 is a candidate relay protein for a subset of ACTH actions in the adrenal cortex.

Adrenal Cortex↗
PubMed1999

[Thrombospondins: multimodular proteins with angiostatic function].

The thrombospondins (TSPs) are a family of multimodular proteins that bind to the extracellular matrix with strong affinity. Of the five members of the TSP family, TSP1 and TSP2 are the only ones that inhibit endothelial cell migration in vitro and neoangiogenesis in vivo. This angiogenesis-inhibiting effect is mediated by interaction of a short sequence in type I modules with the membrane receptor CD36. TSP1 and TSP2 gene knockout experiments in mice showed increased blood vessel density in TSP2 -/- but no such alteration in TSP1 -/- animals. Loss of TSP1 gene expression was correlated with acquisition of an angiogenic phenotype in several models of human malignant tumors. Taken in concert, these findings suggest that TSP1 and, to a lesser extent, TSP2 may have therapeutic potential as angiogenesis-inhibiting factors.

Animals↗
PubMed1998

Smad3 is involved in the intracellular signaling pathways that mediate the inhibitory effects of transforming growth factor-beta on StAR expression.

Transforming growth factor betas (TGFbetas) constitute a family of dimeric proteins that regulate growth and differentiation of many cell types. TGFbeta1 is also a potent autocrine regulator of adrenocortical steroidogenesis. We have recently shown that in primary cultures of bovine fasciculo-reticularis cells, the main target of TGFbeta is the steroidogenic acute relay protein (StAR), a key protein necessary for intramitochondrial cholesterol transport. Here, we show that StAR expression is also inhibited by TGFbeta1 in the human adrenocortical carcinoma cell line NCI-H295R. This inhibitory effect is mediated by Smad proteins. Indeed, we found that overexpression of wild-type Smad3 inhibited endogenous StAR mRNA expression while overexpression of a dominant negative Smad3 protein reversed the inhibitory effect of TGFbeta1 on StAR mRNA expression. Taken together, these results demonstrate that the Smad3 protein is involved in TGFbeta-dependent regulation of steroidogenesis.

Adrenal Cortex↗
PubMed1998

Hormonal regulation of focal adhesions in bovine adrenocortical cells: induction of paxillin dephosphorylation by adrenocorticotropic hormone.

A study of bovine adrenocortical cell shape on adrenocorticotropic hormone (ACTH) challenge showed that the cells round up and develop arborized processes. This effect was found to be (1) specific for ACTH because angiotensin II and basic fibroblast growth factor have no effect; (2) mediated by a cAMP-dependent pathway because forskolin reproduces the effect of the hormone; (3) inhibited by sodium orthovanadate, a phosphotyrosine phosphatase inhibitor, but unchanged by okadaic acid, a serine/threonine phosphatase inhibitor; and (4) correlated with a complete loss of focal adhesions. Biochemical studies of the focal-adhesion-associated proteins showed that pp125fak, vinculin (110 kDa) and paxillin (70 kDa) were detected in the Triton X-100-insoluble fraction from adrenocortical cells. During cell adhesion on fibronectin as substratum, two major phosphotyrosine-containing proteins of molecular masses 125 and 68 kDa were immunodetected in the same fraction. A dramatic decrease in the extent of tyrosine phosphorylation of these proteins was observed within 60 min after treatment with ACTH. No change in pp125fak tyrosine phosphorylation nor in Src activity was detected. In contrast, paxillin was found to be tyrosine-dephosphorylated in a time-dependent manner in ACTH-treated cells. Sodium orthovanadate completely prevented the effect of ACTH. These observations suggest a possible role for phosphotyrosine phosphatases in hormone-dependent cellular regulatory processes.

Adrenal Cortex↗
PubMed1998

Transforming growth factor beta1 decreases cholesterol supply to mitochondria via repression of steroidogenic acute regulatory protein expression.

Transforming growth factor-betas (TGF-betas) constitute a family of dimeric proteins that affect growth and differentiation of many cell types. TGF-beta1 has also been proposed to be an autocrine regulator of adrenocortical steroidogenesis, acting mainly by decreasing the expression of cytochrome P450c17. Here, we demonstrate that TGF-beta1 has a second target in bovine adrenocortical cells, namely the steroidogenic acute regulatory protein (StAR). Indeed, supplying cells with steroid precursors revealed that TGF-beta1 inhibited two steps in the steroid synthesis pathway, one prior to pregnenolone production and another corresponding to P450c17. More specifically, TGF-beta1 inhibited pregnenolone production but neither the conversion of 25-hydroxycholesterol to pregnenolone nor P450scc activity. Thus, TGF-beta1 must decrease the cholesterol supply to P450scc. We therefore examined the effect of TGF-beta1 on the expression of StAR, a mitochondrial protein implicated in intramitochondrial cholesterol transport. TGF-beta1 decreased the steady state level of StAR mRNA in a time- and concentration-dependent manner. This inhibition occurs at the level of StAR transcription and depends on RNA and protein synthesis. It is likely that the TGF-beta1-induced decrease of StAR expression that we report here may be expanded to other steroidogenic cells in which a decrease of cholesterol accessibility to P450scc by TGF-beta1 has been hypothesized.

Animals↗