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G Serrero

Publications and source records attributed to G Serrero.

At least 37 records · Page 2Linked to original sources
PubMed1995

Prostaglandin F2 alpha inhibits epidermal growth factor binding to cellular receptors on adipocyte precursors in primary culture.

Prostaglandin F2 alpha (PGF2 alpha) and epidermal growth factor (EGF) are potent differentiation inhibitors of adipocyte precursors in primary culture. We show here that PGF2 alpha specifically inhibited EGF binding to adipocyte precursors in a dose dependent fashion. Scatchard analysis indicates that PGF2 alpha causes a 50% decrease in the number of available EGF cell surface receptors without change in receptor affinity. Comparison of EGF binding at different temperatures and on fixed cells indicates that PGF2 alpha increases internalization of EGF-receptor complexes in adipocyte precursors. Phorbol myristate acetate (PMA) also inhibited EGF binding in adipocyte precursors. PGF2 alpha effect was abolished in cells exposed to prolonged treatment with PMA indicating that PGF2 alpha effect on EGF binding is mediated by protein kinase C. These results would suggest that in adipocyte precursors PGF2 alpha may be the physiological mediator of phorbol ester effect on EGF receptor properties.

Adipose Tissue↗
PubMed1995

Prostaglandin F2 alpha stimulates transforming growth factor-alpha expression in adipocyte precursors.

Transforming growth factor-alpha (TGF alpha) and prostaglandin F2 alpha (PGF2 alpha) are potent inhibitors of adipocyte differentiation. We demonstrate here that TGF alpha messenger RNA (mRNA) is expressed in freshly isolated fat pads and in primary culture of adipocyte precursors cultivated in defined medium before and after differentiation. We show that PGF2 alpha stimulated TGF alpha mRNA expression in a dose-dependent manner. PGF2 alpha also stimulated TGF alpha production in the culture medium of adipocyte precursors in primary culture. PGF2 alpha stimulated TGF alpha mRNA expression in both undifferentiated and differentiated cells. 9 alpha,11 beta-PGF2 alpha, which also inhibited adipose differentiation, stimulated TGF alpha mRNA expression similarly to PGF2 alpha, whereas other PGs had no effect on TGF alpha mRNA expression. The time-course experiment indicates that the stimulation of TGF alpha mRNA expression by PGF2 alpha is observed within 6 h of exposure to PGF2 alpha and is inhibited by treatment of the cells with actinomycin D. The effect of PGF2 alpha on TGF alpha expression did not require activation of protein kinase C and was fully reversible. As both TGF alpha and PGF2 alpha are inhibitors of adipose differentiation, it is suggested that stimulation of TGF alpha expression by PGF2 alpha could represent an amplification mechanism to modulate adipocyte precursor differentiation and adipocyte function within the adipose tissue.

Adipocytes↗
PubMed1994

Characterization of calcium-independent cytosolic phospholipase A2 activity in the submucosal regions of rat stomach and small intestine.

This study was undertaken to compare the calcium-independent phospholipase A2 (PLA2) activities in the cytosols of twelve rat tissues and to determine whether their activities were distinct. 1-O-Alk-1'-enyl-2-[14C]-oleoyl-sn-glycero-3-phosphocholine (PlsC) and 1-O-Alk-1'-enyl-2-[14C]oleoyl-sn-glycero-3-phosphoethanolamine (PlsE) were synthesized and used as substrates, instead of phosphatidyl compounds, to exclude hydrolysis by cytosolic PLA1 activity that could be present in some of the cytosolic preparations. For each tissue, we examined substrate specificity, pH optimum, and effect of adenosine triphosphate (ATP) and ATP analogues. PLA2 activity was detected in eleven out of the twelve issues examined. Based on substrate specificity and pH optimum, cytosolic calcium-independent PLA2 were classified in three groups. The first group, which included PLA2 from small intestine, stomach and spleen, had the highest specific activity with PlsC as substrate (1253, 309 and 75 nmol/mg protein/hour, respectively) and an optimal pH at 6.5. Activity with PlsE as substrate was much lower (20-37%) than with PlsC. The second group of PLA2 activities included the cytosolic activities from thymus, lung, liver and pancreas that showed lower specific activities for both substrates (14-23 nmol/mg protein/hour with PlsC) and had a broader optimal pH range of 6.1 to 7.5. The cytosols from brain, kidney, heart and muscle comprised the third PLA2 group that was found to have a higher specific activity with PlsE (5-20 nmol/mg protein/hour) than PlsC and an optimal pH range from 7.4 to 7.9.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1993

A novel mouse gene highly conserved throughout evolution: regulation in adipocyte differentiation and in tumorigenic cell lines.

A cDNA clone referred to as 168 was previously isolated from mouse 1246 adipocytes by differential hybridization on the basis of its down regulation in adipocytes when compared to preadipocytes. 5' RACE was used to obtain a full length clone of 761 bp encoding for a highly basic 25 kD polypeptide that is extremely conserved in several diverse species of eukaryotes. There is a single amino acid substitution at position 202 compared to the human homolog, QM, a putative tumor suppressor. Clone 168 mRNA decreases 80% in rat primary culture of adipocytes compared to preadipocytes and does not decrease when differentiation is blocked by PGF2 alpha or EGF, indicating that the decrease is correlated with expression of the differentiation phenotype. Finally, two 1246 cell line variants that exhibit altered growth and increased tumorigenicity have a similar level of 168 mRNA when compared to the non tumorigenic adipogenic parent cell line.

Adipocytes↗
PubMed1993

Purification of an autocrine growth factor homologous with mouse epithelin precursor from a highly tumorigenic cell line.

PC cell line is a highly tumorigenic insulin-independent variant from the teratoma-derived adipogenic cell line 1246. Culture medium of PC cells contains a growth promoting activity for 3T3 cells and producer cells. PC cell-derived growth factor (PCDGF) was purified to homogeneity from PC cell-conditioned medium as an apparent 88-kDa protein by chromatography on heparin-Sepharose, Sephacryl S-200, and phenyl-Sepharose. Digestion with peptide-N-glycosidase F yielded an apparent 68-kDa protein component indicating that PCDGF is a glycoprotein containing about 20 kDa of carbohydrate. Partial sequence from Edman degradation of peptide fragments obtained by digestion of PCDGF with cyanogen bromide and trypsin demonstrates that PCDGF contains regions of sequence identity to that deduced from the granulin or epithelin precursor cDNAs. Granulins are small polypeptides purified from granulocyte extracts with no apparent biological functions. Epithelins are cell growth modulators purified as small molecular mass 6-kDa polypeptides from kidney extracts. The existence of a large molecular mass precursor for granulin or epithelin has been predicted based upon recently cloned cDNAs encoding these biomolecules within a 63.5-kDa protein with putative glycosylation sites. No biological activity has previously been attributed to the precursor. The present results indicate that PCDGF is a potential precursor for epithelin and/or granulin, that this 88-kDa protein is secreted and glycosylated, and that it can function as a mitogen for 3T3 cells as well as an autocrine growth factor for PC cells.

3T3 Cells↗
PubMed1993

Structure of the gene encoding mouse adipose differentiation-related protein (ADRP).

Adipose differentiation-related protein (ADRP) is a novel 50-kDa membrane-associated protein whose message levels are induced rapidly and maximally after triggering adipocyte differentiation. The gene encoding mouse ADRP has been isolated and characterized from four overlapping lambda phage clones. The gene spans 14 kb and contains 8 exons and 7 introns. Exons range in size from 50 to 696 bp and intron sizes range from 87 bp to 4.3 kb. Major and minor transcription initiation sites were determined 76 and 78 bp, respectively, upstream of the initiator methionine. A TATTTTA sequence is centered 30 bp upstream of the major transcription start site, and within the 5'-flanking region there are several putative transcription factor binding sites. ADRP has been mapped to chromosome 4, specifically between the b and Ifa loci. A second ADRP-like gene was isolated and partially characterized. This second locus is not expressed in 12 different mouse tissues and shares 87% sequence similarity to ADRP over exon and intron regions analyzed. Finally, this is the first reported genomic structure of ADRP.

Adipose Tissue↗
PubMed1993

Nucleotide sequence of the C-terminal region of the mouse epidermal growth factor receptor and expression in teratoma-derived cell lines with increased tumorigenic properties.

The isolation of a cDNA corresponding to a portion (amino acid 943 to 1073) of the cytoplasmic domain of the mouse EGF receptor surrounding the auto phosphorylation sites was obtained by using the reverse transcriptase polymerase chain reaction (RT-PCR) approach. Deduced amino acid sequence of mouse EGF receptor (EGFr) shows a 92% and 76% homology to corresponding regions in the human and the chicken EGFr, respectively. This cDNA was used to develop a sensitive RNase protection assay to investigate EGF receptor mRNA expression in mouse C3H teratoma derived cell lines with increased tumorigenic properties which display a progressive decrease of EGF binding and response. The results show that increased tumorigenicity was not accompanied by a change in EGF receptor mRNA expression. Moreover, they indicate that the RNase protection assay developed using the probe described here is a sensitive approach to investigate EGF receptor expression in murine cells.

Amino Acid Sequence↗
PubMed1993

Inhibition of adipose differentiation by 9 alpha, 11 beta-prostaglandin F2 alpha.

Prostaglandin F2 alpha (PGF2 alpha) is a potent adipose differentiation inhibitor for the adipogenic cell line 1246 and for adipocyte precursors in primary culture with an ED50 of 3 x 10(-8) M. In this paper, we examined the effect of several prostaglandins which have structural similarities with PGF2 alpha on the differentiation of 1246 cells and of adipocyte precursors in primary culture. The results show that only 9 alpha,11 beta-PGF2 alpha is as potent as PGF2 alpha to inhibit differentiation of adipocyte precursors in primary culture and of the adipogenic cell line 1246. In the presence of 9 alpha,11 beta-PGF2 alpha, the cells remained fibroblast-like, typical of undifferentiated adipocyte precursors. Triglyceride accumulation and increase of specific activity for glycerol-3-phosphate dehydrogenase were inhibited. In addition, mRNA expression of early markers of differentiation such as lipoprotein lipase (LPL) and fatty acid binding protein (FAB) was decreased. The isomer 9 beta,11 alpha-PGF2 alpha and other PGF2 alpha derivatives were inactive. These results provide new information on the biological activity of 9 alpha,11 beta-PGF2 alpha as an inhibitor of adipose differentiation and about the structural characteristics of prostaglandins required for maintenance of a high adipose differentiation inhibitory effect.

Adipose Tissue↗
PubMed1993

Impaired epidermal growth factor production in genetically obese ob/ob mice.

Epidermal growth factor (EGF) is a potent inhibitor of adipose differentiation in vitro and delays adipose tissue development in vivo. Here we show that in the homozygous male obese mice the level of EGF in the submaxillary gland and plasma is significantly lower than in the glands and plasma of age-matched control littermates. This EGF deficiency in ob/ob mice was observed as early as 5 wk of age when obesity had just become apparent and was also found in adult mice. The level of prepro-EGF mRNA expression in the submaxillary gland was also lower in obese mice than in control littermates. However, the level of kidney prepro-EGF mRNA was the same in mice with both phenotypes, suggesting that the regulation of prepro-EGF mRNA expression is different in both tissues. These results indicate that genetic obesity in mice is accompanied by a decrease in the production of EGF.

Adipose Tissue↗
PubMed1993

Fibroblast growth factor inhibits proliferation of a highly tumorigenic insulin-independent teratoma-derived cell line.

The present paper examines the effect of basic fibroblast growth factor (bFGF) on the proliferation of teratoma-derived cell lines having increased tumorigenic properties isolated from the non-tumorigenic adipogenic cell line 1246. Although FGF is a mitogen for the non tumorigenic 1246 cells and for the moderately tumorigenic 1246-3A cells derived from the 1246 cells, bFGF inhibits the proliferation and DNA synthesis of the highly tumorigenic PC cells starting at concentration as low as 30 pg/ml. The inhibitory effect of FGF on PC cell growth is irreversible as demonstrated by the inability of the cells to resume proliferation once FGF is removed from the culture medium. Comparison of 125I-bFGF binding to the three cell lines was performed. Based on the Scatchard analysis of the binding data, PC cells display only low affinity class of FGF binding sites whereas 1246 and 1246-3A cells presented also high affinity binding sites. The inhibitory effect of FGF on PC cells did not go through activation of a PKC mediated pathway, which is also known to inhibit PC cell proliferation, since FGF inhibition of PC cell growth was still apparent after PKC down regulation. FGF was still able to transiently stimulate the expression of mRNA for early growth associated genes as demonstrated by c-myc and c-fos expression, although it inhibited cell proliferation on PC cells. Our data demonstrate that the highly tumorigenic teratoma cells acquire an inhibitory response for a factor which is growth stimulatory to non-tumorigenic and moderately tumorigenic cells from which they are derived.

Adipose Tissue↗
PubMed1992

Isolation and characterization of a full-length cDNA coding for an adipose differentiation-related protein.

We have previously isolated from a 1246 adipocyte cDNA library a cDNA clone called 154, corresponding to a mRNA that increases abundantly at a very early time during the differentiation of 1246 adipocytes and in adipocyte precursors in primary culture. We show here that the mRNA encoded by this cDNA is expressed abundantly and preferentially in mouse fat pads. A full-length cDNA for clone 154 was isolated by the RACE (rapid amplification of cDNA ends) protocol. Sequence analysis of this cDNA indicates that it encodes a protein of the 425 amino acids [tentatively named adipose differentiation-related protein (ADRP)] that does not have any similarity with sequences contained in the GenBank DNA and Protein Identification Resource protein data bases. Immunoblot of 1246 cell extracts with an antibody raised against the expressed ADRP shows that the 1246 cells contain a 50-kDa protein, the production of which increases as the cells differentiate. Localization of ADRP in 1246 cells indicates that ADRP is absent from nuclear and cytosolic fractions and is found as a membrane-associated protein. These results demonstrate that adipocyte differentiation is accompanied by early expression of a mRNA encoding a membrane-associated adipose differentiation related protein that is adipose tissue specific in vivo.

Adipose Tissue↗
PubMed1992

Effect of testosterone on the growth properties and on epidermal growth factor receptor expression in the teratoma-derived tumorigenic cell line 1246-3A.

1246-3A is an insulin-independent tumorigenic cell line isolated from the C3H mouse teratoma-derived adipogenic cell line 1246. In the present paper, we have demonstrated that testosterone inhibits the in vivo tumorigenic properties of the 1246-3A cells. Castrated male mice receiving injections of 1246-3A cells developed larger tumors at a higher frequency than sham-operated animals. Administration of testosterone to castrated male mice resulted in a dramatic decrease in tumor development. In vitro studies indicated that testosterone inhibited by 50% the proliferation of the 1246-3A cells in culture. However, growth inhibition was observed only if the cells had been cultivated in the presence of testosterone for at least 4 days. In contrast, testosterone had little effect on the proliferation of the parent cell line 1246. Binding of several polypeptide growth factors was examined in cells cultivated in the absence and in the presence of testosterone. Testosterone increased 125I-EGF specific binding to 1246-3A cells. Scatchard analysis of EGF binding indicated that testosterone treatment induced a 2.4-fold increase in the number of cell surface EGF binding sites. This was accompanied by an increase in the intensity of cross-linked EGF receptors on the cells treated with testosterone. In addition, 1246-3A cells cultivated for 9 days in the presence of testosterone displayed a 10-fold increase in the level of EGF receptor mRNA when compared to 1246-3A cells maintained in its absence. Similar to its action on cell proliferation, the increase in EGF receptor number and mRNA expression was observed mainly if 1246-3A cells had been exposed to testosterone for 9 days. The data presented in this paper demonstrate that both in vivo and in vitro, testosterone induces in the teratoma-derived 1246-3A cell line phenotypic changes such as growth inhibition and modulation of EGF receptor expression.

Animals↗
PubMed1992

Prostaglandin F2 alpha inhibits the differentiation of adipocyte precursors in primary culture.

Influence of arachidonate metabolite pathway on adipose differentiation was investigated using primary culture of adipocyte precursors in defined medium. Treatment of the cells with cyclooxygenase inhibitors stimulates adipose differentiation by at least 2-fold. Among the various arachidonate metabolites tested, only prostaglandin F2 alpha (PGF2 alpha) was found to inhibit the differentiation of adipocyte precursors in a dose dependent fashion. Other eicosanoids tested did not have any effect. A 50% inhibition of adipose differentiation was observed with a dose of PGF2 alpha of 3 x 10(-9)M to 7 x 10(-9)M according to the strain of rats used. Maximal inhibition occurred at PGF2 alpha concentrations equal or higher than 10(-8)M. PGF2 alpha inhibited not only the expression of late markers of adipose differentiation such as G3PDH and triglycerides accumulation but also the mRNA expression of early markers of adipose differentiation such as clone 154, lipoprotein lipase and ap2 gene. These results indicate that PGF2 alpha represents a physiological negative modulator of adipose differentiation.

Adipose Tissue↗
PubMed1992

The primary culture of mouse adipocyte precursor cells in defined medium.

1. A defined medium supporting the proliferation and differentiation of adipocyte precursors isolated from inguinal fat pads of 8-12-day-old mice was developed. 2. It consists of a 1:1 mixture of DME and WAJC404A media supplemented with insulin (10 micrograms/ml), transferrin (10 micrograms/ml), fibroblast growth factor (10 ng/ml) and high density lipoproteins (HDL) (90 micrograms protein/ml). 3. DME-F12 medium (1:1 mixture) used as a nutrient mixture in the defined medium of rat and human adipocyte precursors was inadequate for cultivating mouse adipocyte precursors. 4. HDL had a definite beneficial effect on both preadipocyte growth and differentiation. 5. Differentiation was enhanced by addition of dexamethasone (10(-9) M) but could be almost completely inhibited by transforming growth factor beta 1 (TGF-beta 1). 6. TGF-beta 1 was shown to be effective only when present in the early stages of differentiation.

Adipose Tissue↗
PubMed1992

Paracrine regulation of adipose differentiation by arachidonate metabolites: prostaglandin F2 alpha inhibits early and late markers of differentiation in the adipogenic cell line 1246.

The effect of arachidonate metabolites on the differentiation of the adipogenic cell line 1246 was investigated. Among the metabolites examined, only prostaglandin F2 alpha (PGF2 alpha) inhibited differentiation in a dose-dependent fashion with an ED50 of 3 x 10(-9) M. PGF2 alpha inhibited the mRNA expression of lipoprotein lipase, clone 154, and fatty acid-binding protein, which are early markers of differentiation, as well as glycerol-3-phosphate dehydrogenase specific activity and triglyceride accumulation, which are late markers of differentiation. Chronic exposure of 1246 cells to PGF2 alpha before and during differentiation indicated that the cells that have just initiated their differentiation program were the most susceptible to the inhibitory effect of PGF2 alpha. Since 1246 cells produce PGs, we determined whether the PG produced by the cells influenced adipose differentiation. Cyclooxygenase inhibitors added to the culture medium stimulated differentiation of 1246 cells up to 18-fold depending on the type and concentration of inhibitor used. In contrast, lipoxygenase inhibitors had no effect. Treatment of 1246 cells with arachidonic acid resulted in a dose-dependent inhibition of cell differentiation. Oleate or linoleate had no effect. These data indicate that PGF2 alpha inhibits early and late events of adipose differentiation and that the endogenous production of PGs (particularly PGF2 alpha) plays an important role as a negative paracrine or autocrine regulatory pathway of adipose differentiation.

Adipose Tissue↗
PubMed1992

Molecular cloning of a differentiation-related mRNA in the adipogenic cell line 1246.

The 1246 cell line is a C3H mouse teratoma-derived adipogenic cell line that can proliferate and differentiate in defined medium. We have constructed a recombinant phage library containing complementary DNAs (cDNAs) prepared from mRNA of differentiated 1246 cells. This library was screened using a differential hybridization technique. We have isolated five different cDNA clones corresponding to mRNAs that are induced during adipogenesis of 1246 cells and one cDNA clone corresponding to mRNA that is decreased during adipogenesis. Among the mRNAs expressed during adipose differentiation, some are not expressed in undifferentiated cells, whereas some are expressed at very low levels under these conditions. Moreover, the level of induction during differentiation and the temporal expression of the mRNAs corresponding to these cDNAs varied. Our results indicate that one of the cDNA clones isolated, called 154, which selects a 2.2-kilobase mRNA, was induced 100-fold at a very early time during the onset of the differentiation program in 1246 cells and also in adipocyte precursors in primary culture. Direct sequencing of 154 cDNA insert revealed no homology with sequences in GenBank and PIR protein databases. The expression of 154 mRNA was stimulated by accelerators of differentiation such as dexamethasone and isobutylmethylxanthine and inhibited by tumor necrosis factor alpha, transforming growth factor beta, and epidermal growth factor, which are known inhibitors of 1246 cell differentiation. In addition, 154 mRNA level in adipocytes was down-regulated by tumor necrosis factor alpha, but not by transforming growth factor beta or epidermal growth factor. These results suggest that the increase in 154 mRNA expression is related to the onset of adipose differentiation. Further analysis of this clone should allow characterization of a novel protein induced early during the process of differentiation.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Physiological role of epidermal growth factor on adipose tissue development in vivo.

The ability of epidermal growth factor (EGF) to affect adipose tissue development in vivo was investigated. The subcutaneous administration of EGF to newborn NBR rats for 10 days resulted in decreased body weight and fat pad weight that occurred in a dose-dependent fashion. At a dose of 1 micrograms/g of body weight, injected EGF resulted in a 50% decrease of fat pad weight. Although kidney weight was also diminished, the weight of other organs such as liver and intestine remained unchanged or even slightly increased, suggesting that the effect of EGF on fat pad development was not due to a generalized inhibitory action of EGF on the development of the neonate rats. The number of adipocyte precursors in inguinal fat pads of EGF-treated animals was higher than in control animals (1.3-fold for 0.3 micrograms/g of body weight of EGF and 1.8-fold for 1.0 micrograms/g of body weight), whereas the number of mature adipocytes and the amount of triglyceride accumulated per fat pad were concomitantly lower. Adipocyte precursors isolated from EGF-treated animals displayed a reduced differentiation ability in culture and a higher sensitivity to the inhibitory effect of EGF than did cells isolated from control animals. These experiments demonstrate that EGF can retard adipose tissue development in vivo and suggest that EGF plays an important physiological role in the control of adipocyte differentiation.

Adipose Tissue↗