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J M Saez

Publications and source records attributed to J M Saez.

At least 55 records · Page 3Linked to original sources
PubMed1994

Regulation of bovine adrenal cell corticotropin receptor mRNA levels by corticotropin (ACTH) and angiotensin-II (A-II).

Bovine adrenal fasciculata-reticularis cells (BAC) expressed at least four ACTH receptor (ACTH-R) mRNA transcripts, one major of 3.6 kb and three minor of 4.2, 1.8 and 1.3 kb. ACTH and A-II increased ACTH-R mRNA levels in a time- and dose-dependent manner. At maximal concentrations, ACTH caused a 2.7-fold increase in the level of the major transcript of 3.6 kb with an ED50 = 10(-11) M and A-II produced a 2.4-fold increase with an ED50 = 5 x 10(-8) M. Under our experimental conditions, the stimulatory effects of both hormones appeared to be due to post-transcriptional changes rather than to transcriptional regulation since the hormonal effects were also observed in actinomycin-treated cells. The results indicate that regulation of ACTH-R mRNA levels may be one mechanism by which ACTH and A-II regulate adrenocortical functions.

Adrenocorticotropic Hormone↗
PubMed1994

Characterization of the transcription start site of the ACTH receptor gene: presence of an intronic sequence in the 5'-flanking region.

Corticotropin (ACTH) regulates glucocorticoid production through specific receptors on the adrenal cortex. Analysis of the ACTH receptor mRNA in human adrenal has revealed the presence of five transcripts ranging from 1.8 to 11 kilobases (kb). Characterization of the 5'-untranslated regions (UTRs) of the ACTH receptor mRNA demonstrated the presence of one major initiation site of transcription 177 bp away from the ATG codon. Analysis of this 5' sequence showed a perfect alignment with the previously described genomic sequence until position -128 bp from the ATG. The upstream 49-bp sequence was divergent, suggesting the occurrence of a splicing and indicating the presence of an intronic sequence in the UTRs, as well as the presence of an upstream exon containing this 49-bp sequence and located at least 1.8 kb away from the exon encoding the protein.

Adrenal Glands↗
PubMed1994

Regulation of corticotropin and steroidogenic enzyme mRNAs in human fetal adrenal cells by corticotropin, angiotensin-II and transforming growth factor beta 1.

Using cultured human fetal adrenal cells, we have investigated the basal secretion of cortisol and dehydroepiandrosterone sulfate (DHAS) and the effect of corticotropin (ACTH), angiotensin-II (A-II) and transforming growth factor beta 1 (TGF beta 1) on the secretion of these steroids and on the mRNA levels of ACTH receptor (ACTHR), cytochrome P-450scc (cholesterol side-chain cleavage), P450 17 alpha (17 alpha-hydroxylase/17-20 lyase) and 3 beta-HSD (3 beta-hydroxysteroid dehydrogenase). The basal DHAS/cortisol ratio declined progressively between 12.5 and 21 weeks. ACTH treatment enhanced the secretion of cortisol and to a lesser extent that of DHAS, and increased the steroidogenic response to an acute stimulation with ACTH. These changes were associated with increased mRNA levels of ACTHR and of the steroidogenic enzymes. A-II treatment also increased the secretion of both DHAS and cortisol, but less than ACTH, enhanced the responsiveness to ACTH and increased ACTHR, P450scc and P450 17 alpha mRNA levels. In contrast, TGF beta 1 alone or together with ACTH decreased DHAS secretion, but not cortisol secretion. Moreover, TGF beta 1 had no effect on ACTHR and P450scc mRNA levels, decreased by about 50% the mRNA levels of P450 17 alpha both in the absence or presence of ACTH, but enhanced the stimulatory effects of ACTH on 3 beta-HSD mRNA. These results, along with those previously reported, suggest that both A-II and TGF beta may play a role in fetal adrenal function. In addition, they show that the effects of both peptides are qualitatively different from, even sometimes opposite to, those previously reported in bovine and ovine adrenal cells.

3-Hydroxysteroid Dehydrogenases↗
PubMed1994

Peptide hormone and growth factor regulation of nuclear proto-oncogenes and specific functions in adrenal cells.

Among the large number of immediate early genes, nuclear proto-oncogenes of the Fos and Jun families, have been postulated to be involved in the long-term effects of several growth factors on cell differentiation and/or multiplication. Since adrenal cell differentiated functions appear to be regulated by specific hormones and growth factors, the effects of these factors on proto-oncogene mRNA levels were analysed in bovine adrenal fasciculata cells (BAC) in culture. Corticotropin (ACTH) and insulin-like growth factor I increased c-fos and jun-B mRNA, but had no effect on c-jun mRNA and these early changes were associated with a later increase in BAC specific function [ACTH receptors, cytochrome P450 17 alpha) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD)] and an enhanced steroidogenic responsiveness to both ACTH and angiotensin-II (A-II). On the other hand, A-II increased the three proto-oncogene (c-fos, c-jun and jun-B) mRNAs, induced a decrease of P450 17 alpha and 3 beta-HSD and caused a marked homologous and heterologous (ACTH) densitization. Transforming growth factor beta 1 which only increased jun-B mRNA, markedly reduced BAC differentiated functions and the steroidogenic responsiveness to both ACTH and A-II. Thus, it is postulated that the proto-oncoproteins encoded by the immediate early genes may play a role in the long-term effects of peptide hormones and growth factors on BAC differentiated functions.

Adrenal Glands↗
PubMed1994

Regulation of adrenal cell-differentiated functions by growth factors.

Adrenal cell-differentiated functions and, therefore, their steroidogenic capacity can be regulated in an opposite direction by insulin-like growth factor I (IGF-I) and transforming growth factor-beta 1 (TGF beta 1). The enhanced steroidogenic responsiveness of bovine and ovine adrenal cells treated with IGF-I can be explained by its positive effects on the number of corticotropin (ACTH) and angiotensin II (A-II) receptors associated with an increase in the alpha s and alpha i subunits of G proteins but also by its effects on several steps of the steroidogenic pathway. In contrast, TGF beta 1 is a potent inhibitor of differentiated functions of both bovine and ovine adrenal cells. TGF beta 1 reduces ACTH and A-II receptor number, inhibits cAMP formation, and decreases several steroidogenic enzyme activities. The physiological role of these peptides in adrenal cells is strengthened by the fact that both are synthetized and secreted by these cells and that their secretion can be regulated by the specific hormones, ACTH and A-II.

Adrenal Glands↗
PubMed1994

Regulation of corticotropin receptor number and messenger RNA in cultured human adrenocortical cells by corticotropin and angiotensin II.

The regulation of ACTH receptor binding sites and mRNA by ACTH and angiotensin II (A-II) was studied using cultured human adrenal fasciculata reticularis cells (HAC). These cells expressed two major ACTH receptor transcripts of 1.8 and 3.4 kb and three minor ones of 4, 7, and 11 kb. ACTH increased the levels of all these transcripts in a time- and dose-dependent manner. At a maximal concentration of 10(-8) M, ACTH enhanced 21- and 4-fold the level of ACTH receptor mRNA and the number of receptors per cell, respectively. Pretreatment of HAC with A-II produced a dose-dependent enhancement of ACTH receptor mRNA that was associated with an increase of both ACTH receptor number and responsiveness to this hormone. The effects of A-II were completely blocked by an AT1 receptor subtype antagonist but not by an AT2 antagonist. The effects of ACTH together with A-II on ACTH receptor mRNA were greater than those induced by each hormone alone. These results show that ACTH receptor number and mRNA are positively regulated by the two main hormones (ACTH and A-II) which, in vivo, regulate adrenocortical functions. In addition, they also show that HAC are a target for A-II. Thus, regulation of ACTH receptors may be one mechanism by which ACTH and A-II regulate adrenocortical functions under both normal and pathological conditions.

Adrenal Cortex↗
PubMed1994

Expression and regulation of transforming growth factor-beta 1 messenger ribonucleic acid and protein in cultured porcine Leydig and Sertoli cells.

In the present work, the expression and secretion of transforming growth factor-beta 1 (TGF beta 1) by immature pig Leydig and Sertoli cells were investigated. Both cell types express two TGF beta 1 mRNA transcripts of 2.5 and 3.5 kilobases, and the levels were 2.6-fold higher in Leydig than in Sertoli cells. In the latter cells, mRNA levels were enhanced when cultured cells were stimulated by epidermal growth factor and phorbol ester (4-beta-phorbol 12-myristate 13-acetate) and significantly decreased by FSH and testosterone. Using a polyclonal antibody raised against a synthetic peptide that corresponded to the carboxyl-terminal region of TGF beta 1 and recognized this peptide, but not TGF beta 2 or TGF beta 3, specific immunostaining of both Leydig and Sertoli cells was demonstrated in situ, after cell isolation, and during culture. The immunostaining was more marked in Leydig cells than in Sertoli cells. Western blot analysis of Leydig or Sertoli cell-conditioned medium demonstrated a band of 25 kilodaltons, which was shifted to 12.5 kilodaltons under reducing conditions. Using the mink lung epithelial cell bioassay for TGF beta 1, we could demonstrate the presence of TGF beta 1-like activity in Leydig and Sertoli cell-conditioned media after acid treatment, but not before activation. The inhibitory effects of both pure TGF beta 1 and acidified conditioned medium were almost completely blunted by the TGF beta 1 antibody. The amounts of TGF beta 1 secreted by Sertoli and Leydig cells were not significantly different and varied between 400-800 pg/48 h.10(6) cells. These studies demonstrate for the first time that both pig Leydig and Sertoli cells express TGF beta 1 mRNA, and the TGF beta 1-like activity secreted by these cells corresponds to TGF beta 1. As TGF beta 1 has been demonstrated to have strong effects on testicular cells, in particular on Leydig cell functions, it is suggested that local secreted TGF beta 1 may play a role in the autocrine/paracrine regulation of testicular functions.

Animals↗
PubMed1994

Regulation by growth factors of angiotensin II type-1 receptor and the alpha subunit of Gq and G11 in bovine adrenal cells.

In the present work we have investigated the effects of several growth factors on the expression of Angiotensin II (A-II) receptors subtype AT1 and their pertussis toxin-insensitive coupling to G-proteins in bovine adrenal fasciculata-reticularis cells (BAC). Insulin, Insulin-like growth factor and basic Fibroblast growth factor increased AT1 receptors (mRNA and binding sites) as well as the alpha subunit of Gq (mRNA and protein) and G11 (protein). These changes were associated with an enhanced A-II-induced inositol phosphate accumulation and cortisol production. In contrast, Transforming growth factor beta 1, which reduced slightly AT1 binding sites, but not the level of alpha q or alpha 11 proteins, did not change the A-II-induced inositol phosphate accumulation. However, this factor, as previously reported, markedly reduced cortisol production.

Adrenal Glands↗
PubMed1994

Human cultured adrenal fasciculata-reticularis cells are targets for angiotensin-II: effects on cytochrome P450 cholesterol side-chain cleavage, cytochrome P450 17 alpha-hydroxylase, and 3 beta-hydroxysteroid-dehydrogenase messenger ribonucleic acid and proteins and on steroidogenic responsiveness to corticotropin and angiotensin-II.

In the present study we have examined the effects of ACTH and angiotensin-II (A-II) on cultured human adult fasciculata-reticularis-specific functions. When cells were cultured in a chemically defined medium, the mRNA levels of cholesterol side-chain cleavage enzyme (P450scc), 17 alpha-hydroxylase/17,20-lyase (P45017 alpha), and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) progressively declined, so that by day 6 of culture, less than 20% of those observed in freshly isolated cells were present. ACTH and A-II, in a dose- and time-dependent manner, enhanced the mRNA levels of the three enzymes and the protein levels of P45017 alpha and 3 beta HSD, but not protein levels of P450scc. At maximal concentrations, the effects of ACTH on P450scc mRNA levels and P45017 alpha mRNA and protein levels were significantly greater than those induced by A-II, but the effects of both hormones on 3 beta HSD mRNA and protein were similar. At maximal concentrations, the effects of ACTH and A-II were additive only on 3 beta HSD mRNA and protein. The cortisol production of cells pretreated with ACTH or A-II was significantly higher than that of control cells, but the effects of ACTH were greater than those of A-II. Moreover, the acute steroidogenic responses to ACTH or A-II of cells pretreated with either hormone, were significantly higher than those of control cells. In conclusion, the present results demonstrate that human adult adrenal fasciculata-reticularis cells are targets for A-II, because 1) A-II acutely stimulates cortisol secretion and causes a long term increase in P450scc, P45017 alpha, and 3 beta HSD mRNA levels; 2) the steroidogenic responsiveness of A-II-pretreated cells to both ACTH and A-II was increased; and 3) the positive effects of A-II alone or in association with ACTH on steroidogenic enzyme gene expression are opposite those previously reported on bovine and ovine adrenal cells.

3-Hydroxysteroid Dehydrogenases↗
PubMed1994

Effects of transforming growth factor-beta 1 on human adrenocortical fasciculata-reticularis cell differentiated functions.

Transforming growth factor-beta 1 (TGF beta 1) has been reported to have a strong inhibitory effect on the specific function of adrenal cells of several species. In the present study, we examined the effects of TGF beta 1 on cultured human fasciculata-reticularis cells. TGF beta 1 alone had no effect on ACTH receptor messenger ribonucleic acid (mRNA) levels and was unable to reduce the strong stimulatory effects of ACTH on its own receptor. However, TGF beta 1 enhanced angiotensin-II type 1 receptor mRNA and binding sites. Treatment with TGF beta 1 increased significantly the levels of 3 beta-hydroxysteroid dehydrogenase mRNA, reduced those of cytochrome P-450 17 alpha-hydroxylase mRNA, and had no effect on cholesterol side-chain cleavage cytochrome P-450 mRNA. Whatever the experimental condition, TGF beta 1 did not reduce basal or ACTH-stimulated cortisol production, but the production of dehydroepiandrosterone sulfate of TGF beta 1-treated cells was always decreased. The effects of TGF beta 1 on 3 beta-hydroxysteroid dehydrogenase mRNA and dehydroepiandrosterone sulfate were opposite the change observed at the time of adrenarche. As adrenal cells express TGF beta 1 mRNA, it is tempting to postulate that a local diminution of TGF beta 1 might be involved in adrenarche. Our findings also illustrate the specific species differences and, therefore, the caution to extrapolate to humans the results observed in other species.

Adrenal Cortex↗
PubMed1994

[Functional interactions between somatic cells in the male gonad].

The existence of multiple interactions between somatic cells in the testis, has been suspected in vivo, and demonstrated in vitro by cocultures and cultures in the presence of conditioned media. Long term Sertoli-Leydig cell interactions results in a local regulation of the specific differentiated functions of these cells, and more precisely in a control of their responsiveness to their respective gonadotropin. Among the paracrine and/or autocrine factors, growth factors are involved in the control of differentiated functions. It is the case for IGF-I and TGF-beta, which respectively exhibit a stimulatory and inhibitory effect on Leydig cell functions and more specifically on LH responsiveness.

Cell Communication↗
PubMed1993

Regulation by growth factors (IGF-I, b-FGF and TGF-beta) of proto-oncogene mRNA, growth and differentiation of bovine adrenocortical fasciculata cells.

Nuclear proto-oncoproteins have been implicated in the regulation of gene expression by peptidic hormones and growth factors during cell proliferation and differentiation. In the present study we have investigated in bovine adrenal cells (BAC) the effects of basic fibroblast growth factor (b-FGF), insulin-like growth factor 1 (IGF-I) and transforming growth factor beta (TGF-beta) on c-jun, jun-B and c-fos mRNA levels and on cell growth and differentiation. Factors able to enhance the three proto-oncogenes (IGF-I, b-FGF and angiotensin II (A-II)) stimulate cell growth, whereas those inhibiting cell growth (TGF-beta and ACTH) decrease c-jun mRNA level. These results suggest that expression of c-jun may be required to induce cell proliferation. The relation between proto-oncogenes and the expression of differentiated functions appears to be more complex. Whereas IGF-I, b-FGF and A-II increase the three nuclear proto-oncogenes, the effects of IGF-I and b-FGF on cytochrome P450 17 alpha-hydroxylase mRNA levels are opposite to those of A-II. On the other hand, while TGF-beta and A-II have inhibitory effects on P450 17 alpha mRNA level, they have opposite effects on c-jun mRNA.

Animals↗
PubMed1993

Characterization and regulation of the angiotensin II type-1 receptor (binding and mRNA) in human adrenal fasciculata-reticularis cells.

The classical concept of human adrenal physiology indicates that only glomerulosa cells are the target of A-II. Herein, we demonstrated that cultured human adrenal fasciculata-reticularis cells were also responsive to this hormone. Indeed, these cells contained high affinity (Kd = 0.9-1.1 nM) and low capacity (8,000-13,000 sites/cell) A-II receptors, and more than 95% of them were of the type-1. These AT1 receptors are functional since A-II was able to increase cortisol production after 48 h of treatment. These effects were inhibited by losartan, an AT1 antagonist, but not by CGP42112A, an AT2 antagonist. The expression of the type-1 A-II receptor mRNA was detected in the whole adrenal in both adult and fetus, and in cultured human adrenal fasciculata-reticularis cells. In these cells A-II negatively regulated AT1 receptor mRNA, and this effect was also mediated through the AT1 receptor subtype.

Adult↗
PubMed1993

Identification and characterization of corticotropin receptors in bovine and human adrenals.

Corticotropin (ACTH) receptors have been characterized by covalent cross-linking of radiolabeled ACTH ([125I]ACTH) with the bifunctional cross-linking reagent disuccinimidyl suberate to cultured bovine adrenal fasciculata reticularis cells (BAC), and to crude plasma membrane fractions prepared from both human and bovine adrenals. Incubation of BAC with [125I]ACTH at 20 degrees C followed by cross-linking resulted in the specific labeling of two binding proteins with apparent M(r) of 154,000 and 43,000 as measured by SDS-PAGE under reducing and non-reducing conditions. In addition, in some experiments another band with an apparent M(r) of 124,000 was observed. All of these bands disappeared when the incubation was performed in the presence of an excess of unlabeled ACTH. When BAC were incubated with [125I]ACTH in the presence of 100 microM phenylarsine oxide at 20 degrees C, a condition which prevents the internalization of the ACTH-receptor complex, the bulk of the radioactivity was present in the 43,000 band. After [125I]ACTH cross-linking to BAC, subcellular preparations followed by SDS-PAGE revealed that the 20,000 g fraction contained mainly the 43,000 M(r) form. Cross-linking of [125I]ACTH to plasma membrane-enriched fractions prepared from human and bovine adrenals resulted only in the labeling of the 43,000 protein. These results indicate that the ACTH receptor present at the cell surface is a macromolecule of 43,000, and suggest that the 154,000 form probably represents association of the ACTH-receptor complex to another macromolecule. The 154,000 protein would be formed during or after internalization of the ACTH-receptor complex.

Adrenocorticotropic Hormone↗
PubMed1993

Enhancement of testosterone secretion by normal adult human Leydig cells by co-culture with enriched preparations of normal adult human Sertoli cells.

An in-vitro method was developed to study Sertoli-Leydig cell interactions in man, using testes removed at the time kidneys were removed for transplantation from 6 young adult men (aged 17-45 years) after cerebral death. After collagenase digestion of testicular tissue, Leydig cells were purified on discontinuous Percoll gradients. Two fractions of Leydig cells, 'L2' and 'L3' which differed in their buoyant density (1.05 g < L2 < 1.06 g and 1.06 g < L3 < 1.08 g), were obtained. The Sertoli cell-enriched preparation was obtained from seminiferous tubular fragments after sequential treatment with glycine buffer to remove peritubular-myoid cells, a second collagenase digestion, mechanical fragmentation and washes to remove germ cells. Purified Leydig cells were then cultured either alone or together with Sertoli cells in culture dishes coated with collagen, fibronectin and laminin in a chemically defined medium without serum. The influence of co-culture on basal testosterone secretion was examined in 3 successive 48 h periods.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Stimulatory effect of follicle-stimulating hormone on basal and luteinizing hormone-stimulated testosterone secretions by the fetal rat testis in vitro.

The in vitro effect of FSH on testosterone secretion by the fetal rat testis was studied. Testes were cultured in the presence or absence of either commercial human (h) FSH (Metrodine; 200 mIU/ml) or recombinant hFSH (200 mIU/ml) for 3 days and with 100 ng/ml ovine LH during the last 4 h of culture. To avoid a stimulatory effect by the 0.4% LH that contaminates Metrodine, the cultures were performed in the presence of a monoclonal anti-hLH beta antibody and with a concentration of Metrodine that had no short term stimulatory effect on testosterone production by the fetal testes in vitro. Metrodine treatment had a positive long term effect on both basal and LH-stimulated testosterone secretion by fetal testes explanted on days 18.5, 20.5, and 22.5 postconception, which was abolished by the addition of a monoclonal anti-hFSH beta antibody. LH-free recombinant FSH also augmented basal and LH-stimulated testosterone secretion of testes explanted on days 13.5, 14.5, and 18.5 postconception. The positive effect of recombinant hFSH appeared during the second day of treatment with day 14.5 and 18.5 testes and on the third day of treatment with day 13.5 testes. As it is widely accepted that FSH receptors are exclusively localized on Sertoli cells, these results suggest that on or before day 15.5 of fetal life, 1) Sertoli cells are able to respond to FSH, 2) Sertoli cells can produce factors that are able to act on Leydig cell function, and 3) Leydig cells are sensitive to FSH-induced Sertoli cell factors. In conclusion, this study points out a potential paracrine control of fetal Leydig cell function and/or differentiation by fetal Sertoli cells as soon as fetal Leydig cells differentiate.

Animals↗