In vivo footprinting of the carbamoylphosphate synthetase I cAMP-response unit indicates important roles for FoxA and PKA in formation of the enhanceosome.

The expression of carbamoylphosphate synthetase-I (CPS), the first and rate-determining enzyme of the urea cycle, is regulated at the transcriptional level by glucocorticoids and glucagon, the latter acting via cyclic AMP (cAMP). The hormonal response is mediated by a distal enhancer located 6.3 kb upstream of the transcription-start site. Within this enhancer, a cAMP-response unit (CRU) is responsible for mediating cAMP-dependent transcriptional activity. The CPS CRU contains binding sites for cAMP-response element (CRE)-binding protein (CRE-BP), forkhead box A (FoxA), CCAAT/enhancer-binding protein (C/EBP), and an unidentified protein P1. To gain insight in the protein-DNA interactions that activate the CPS CRU in living cells, we have employed in vivo footprinting assays. Comparison of the fibroblast cell line Rat-1 and the hepatoma cell lines FTO-2B and WT-8 showed that FoxA binds the CPS CRU constitutively in CPS-expressing cells only. Comparison of FTO-2B and WT-8 hepatoma cells, which only differ in cAMP responsiveness, demonstrated that the binding of the other transcription factors is dependent on cAMP-dependent protein kinase (PKA) activity. Finally, we observed a footprint between the CRE and the P1-binding site in the in vivo footprint assay that was not detectable by in vitro footprint assays, implying a major change in CRU-associated chromatin conformation upon CRU activation. These findings indicate that activation of the CRU is initiated in a tissue-specific manner by the binding of FoxA. When cellular cAMP and glucocorticoid levels increase, CRE-BP becomes activated, allowing the binding of the remaining transcription factors and the transactivation of the CPS promoter.

A stat-responsive element in the promoter of the episialin/MUC1 gene is involved in its overexpression in carcinoma cells.

The mucin-like glycoprotein episialin (MUC1) is highly overproduced by a number of human carcinomas. We have shown previously in a variety of mammalian cell lines that overexpression of this very large transmembrane molecule diminishes cellular adhesion, suggesting that episialin/MUC1 overexpression may play an important role in tumor invasion and metastasis. By using in situ hybridization, we show here that episialin/MUC1 mRNA expression can be increased more than 10-fold in breast carcinoma cells relative to the expression in adjacent normal breast epithelium. In search of the molecular mechanism of this overexpression, we observed that the episialin/MUC1 promoter contains a candidate binding site for transcription factors of the STAT family approximately 500 base pairs upstream of the transcription start site. Cytokines and/or growth factors such as interleukin-6 or interferon-gamma can activate STATs. In the human breast carcinoma cell line T47D, both compounds are able to stimulate transcription of a luciferase reporter gene under the control of a 750-base pair MUC1 promoter fragment proximal to the transcription start site. The observed increase is entirely mediated by the single STAT-binding site, since mutation of this site abolishes stimulation of the reporter by interleukin-6 and interferon-gamma. In addition, mutation of the STAT site also decreased the promoter activity in nonstimulated T47D cells, suggesting that the STAT-binding site is among the elements that are involved in the overexpression of MUC1 in tumor cells.

The effect of 9-cis-retinoic acid on proliferation and differentiation of a spermatogonia and retinoid receptor gene expression in the vitamin A-deficient mouse testis.

Retinoid X receptors (RXRs) are key regulators in retinoid signaling. Knowledge about the effects of 9-cis-retinoic acid (9-cis-RA), the natural ligand for the RXRs, may also provide insight in the functions of RXRs. In this study, the effect of 9-cis-RA on spermatogenesis in vitamin A-deficient (VAD) mice was examined. Administration of 9-cis-RA stimulated the differentiation and subsequent proliferation of the growth-arrested A spermatogonia in the testis of VAD mice. However, compared with all-trans-retinoic acid (ATRA), relatively higher doses of 9-cis-RA were necessary. This could not simply be due to a lower or delayed activity of 9-cis-RA, as simultaneous administration of ATRA and 9-cis-RA did not cause a synergistic effect. Instead, the presence of 9-cis-RA diminished the effect of ATRA by approximately one third. Studies of in vivo transport and metabolism showed that ATRA and 9-cis-RA, after administration to VAD mice, penetrated the testis equally well. However, 9-cis-RA was metabolized much faster than ATRA, and other metabolites were formed. This may account for the above-described differential effects of ATRA and 9-cis-RA on spermatogenesis. Similar to ATRA, 9-cis-RA transiently induced the messenger RNA expression of the nuclear RA receptor RAR beta, suggesting a role for this receptor in the effects of retinoids on the differentiation and proliferation of A spermatogonia. In contrast, the messenger RNA expression of the nuclear retinoid receptors RXR alpha, -beta, and -gamma was not changed significantly by administration of their ligand, 9-cis-RA. Hence, 9-cis-RA does not seem to exert its effect on spermatogenesis through altered expression of the RXRs.

Differential expression pattern of retinoid X receptors in adult murine testicular cells implies varying roles for these receptors in spermatogenesis.

Retinoids have previously been shown to be crucial for normal spermatogenesis. The role of retinoic acid receptors has been studied, but relatively little is known about the function of retinoid X receptors (RXRs). To gain more insight in the function of RXRs during spermatogenesis, the cellular localization of RXRs in the mouse testis was examined using immunohistochemistry and RNase protection assays. In both normal and vitamin A-deficient (VAD) testes, a strong immune response to an RXRalpha antibody occurred in Leydig cells, peritubular myoid cells, and A spermatogonia. Weaker signals were found in spermatocytes and spermatids. In normal testes, an RXRbeta antibody gave a reaction in Leydig cells, and, to a lesser extent, in Sertoli cells, A spermatogonia, pachytene spermatocytes, and spermatids. In Leydig cells, a cytoplasmatic signal was found in addition to the nuclear signal. In the VAD testis, only Leydig cells and A spermatogonia were positive, which indicates that RXRbeta expression may be dependent on the retinoid status. Previous studies have shown RXRgamma mRNA expression in the mouse testis at a low level. Nevertheless, an RXRgamma antibody caused a strong immune response in interstitial cells and in A spermatogonia, and a weak signal in pachytene spermatocytes. These immunohistochemical data were supported by the results of RNase protection assays on mRNA of testicular cell isolations. In conclusion, the different RXRs in the mouse testis have distinct expression patterns, suggesting that they may have different functions.

Isolation and characterization of all-trans-retinoic acid-responsive genes in the rat testis.

By way of differential screening of testis cDNA libraries from vitamin A-deficient (VAD) rats before and after administration of all-trans retinoic acid (ATRA), genes, the transcription of which was influenced by ATRA, were isolated. Most clones with an increased transcription encoded different subunits of the same mitochondrial protein complex, cytochrome c oxidase (COX). The mRNA expression of COX increased by a factor 3.9 +/- 1.5 (mean +/- SD, n = 4). This increased expression seems to reflect an increased energy demand in the ATRA-supplemented VAD testis. Also, one gene was isolated, the transcription of which was reduced to about 70% by ATRA. This gene, sulfated glycoprotein 2 (Sgp-2), is a major secretion product of Sertoli cells, the function of which is still unknown. The effect of ATRA on Sgp-2 expression may be direct, since the promoter of Sgp-2 contains a putative ATRA-responsive element (RARE).

Effect of retinoid status on the messenger ribonucleic acid expression of nuclear retinoid receptors alpha, beta, and gamma, and retinoid X receptors alpha, beta, and gamma in the mouse testis.

The testicular gene expression of the retinoic acid receptors, RAR alpha, -beta, and -gamma, was studied in normal mice and in vitamin A-deficient mice after the administration of all-trans-retinoic acid (ATRA). All three types of RARs were expressed in normal and/or vitamin A-deficient testes. Only the expression of RAR beta messenger RNA was transiently induced within 24 h after ATRA injection. ATRA-induced RAR beta expression was also found in purified Sertoli cells, suggesting that these cells mediate at least part of the effect of retinoids on germ cells. When an equimolar amount of retinol was administered instead of ATRA, no induction of RAR beta was seen at the point of maximal induction by ATRA, suggesting that the effect of retinol was delayed and probably less. The related nuclear receptors, RXR alpha, -beta, and, for the first time, gamma, were also shown to be present in the mouse testis. Upon administration of ATRA, messenger RNA expression of RXR alpha and -beta did not change significantly. The expression of RXR gamma was too low to allow quantification. Finally, the effect of the retinoid metabolism inhibitor liarozole on ATRA-induced proliferation of A spermatogonia was examined. The labeling index of A spermatogonia, 24 h after the administration of 0.25 mg ATRA, was significantly lowered by liarozole due to a shift of the maximal 5-bromo-deoxyuridine incorporation to an earlier point (20 h). This indicates that liarozole delays retinoid metabolism, thereby increasing the actual ATRA concentration, and more importantly, that ATRA by itself is an active retinoid in spermatogenesis. Apparently, ATRA does not need to be metabolized to 4-oxo-RA, which was previously shown to be a more potent inducer of spermatogonial proliferation than ATRA, to be effective.

Isolation of the synchronized A spermatogonia from adult vitamin A-deficient rat testes.

A method for isolating A spermatogonia from the adult vitamin A-deficient (VAD) rat testis is described. After removal, the testes were decapsulated and tubules were dissected. An enzymatic digestion with collagenase, hyaluronidase, and trypsin was performed first to eliminate most of the interstitial cells. A second digestion with collagenase and hyaluronidase was performed to obtain a cell suspension with a high number of A spermatogonia. The cell suspension was further enriched with A spermatogonia by preplating on peanut agglutinin and separating on a discontinuous Percoll gradient. By this procedure, purification of the suspension to 70-90% A spermatogonia was obtained. In the seminiferous tubules of the VAD rats, only Sertoli cells, A spermatogonia, and some preleptotene spermatocytes are present. In our rats, the A spermatogonia are almost all arrested in the G1 phase of the cell cycle before the S phase of A1 spermatogonia, and presumably before their differentiation into A1 spermatogonia. After administration of vitamin A, spermatogenesis starts synchronously from these A spermatogonia. The isolation of these synchronized A spermatogonia opens ways to investigate the regulation of differentiation and proliferation of A spermatogonia and the biochemical characteristics of the subsequent types of A spermatogonia.

All-trans-4-oxo-retinoic acid: a potent inducer of in vivo proliferation of growth-arrested A spermatogonia in the vitamin A-deficient mouse testis.

Vitamin A deficiency leads to an arrest of spermatogenesis and a loss of advanced germ cells in male mice. In the present study, the effects of several retinoids and carotenoids on these mouse testis were investigated. First, the proliferative activity of the growth-arrested A spermatogonia in vitamin A-deficient (VAD) mice testis was determined, 20, 24, or 28 h after administration of 0.5 mg all-trans-retinoic acid (RA). The bromodeoxy-uridine (BrdU) labeling index of A spermatogonia in control VAD testis was 5 +/- 1% (n = 4, mean +/- SD). When RA was injected (ip), the highest labeling index was found 24 h after RA administration; 49 +/- 5%. When various concentrations of RA, all-trans-4-oxo-retinoic acid (4-oxo-RA) or all-trans-retinol acetate (ROAc), ranging from 0.13-1 mg, were injected, the labeling index of A spermatogonia always increased in comparison with the VAD situation. A maximum index at 24 h was found when 0.5 mg 4-oxo-RA was injected: 56 +/- 3%. This labeling index was even higher than those after injection of RA or ROAc, 49 +/- 5% and 34 +/- 6% respectively. The increase of the BrdU labeling index was dose dependent. After an initial increase of the labeling indices with increasing retinoid doses, the labeling indices decreased at a higher concentration. This decrease is likely due to a concentration dependent timeshift of the optimum of BrdU labeling to shorter time intervals after retinoid administration because a labeling index of 66 +/- 1% was found 20 h after injection of 1 mg RA. At 24 h, this labeling index was halved: 33 +/- 2%. These indices show that the degree of synchronization of spermatogenesis is also dependent on the retinoid dose. When the dimers of RA and 4-oxo-RA, respectively beta-carotene (beta C) and canthaxanthin, were given, 24 h after administration BrdU-labeling indices comparable with the VAD value were found. Repeated injection of beta C twice a week did induce a reinitiation of spermatogenesis, but compared with RA, the activity of beta C was lower and delayed. It is concluded that 4-oxo-RA is active in adult mammals in vivo. It is at least as potent as RA in the induction of the differentiation and subsequent proliferation of growth-arrested A spermatogonia in VAD mice testis. Furthermore, the degree of synchronization of spermatogenesis is influenced by the retinoid dose. Finally, carotenoids were shown to act in the induction of spermatogonial cell proliferation too but with a lower and delayed activity.

Characteristics of A spermatogonia and preleptotene spermatocytes in the vitamin A-deficient rat testis.

The proliferative activity and other characteristics of germ cells in the vitamin A-deficient (VAD) rat testis were investigated. In the VAD testis, A spermatogonia and preleptotene spermatocytes were found. The A spermatogonia in the VAD testis showed a bromodeoxyuridine (BrdU) labeling index of 6.6 +/- 1.1% and a mitotic index of 2.8 +/- 0.5%. After continuous labeling with BrdU for up to four days, the ultimate labeling index of A spermatogonia was 11.6 +/- 2.5%, which is less than expected. It is concluded that in the VAD rat testis, many of the proliferating A spermatogonia degenerate. During the first 18 h after administration of vitamin A, no increase was observed in either the labeling index or the mitotic index of the A spermatogonia. However, after 24 h the first wave of A spermatogonia in S phase was found, and the first wave in mitosis was found after 48 h. Furthermore, in the VAD testis the DNA content of most of the A spermatogonia was similar to that of Sertoli cells, i.e., 2n. It is concluded that in the VAD situation, nearly all A spermatogonia are arrested before the S phase of the A1 spermatogonia. The hypothesis is put forward that in the VAD testis, the remaining A spermatogonia are the undifferentiated spermatogonia that are unable to differentiate into A1 spermatogonia. The preleptotene spermatocytes in the VAD testis showed a BrdU labeling index of 20.3 +/- 3.5%, while the DNA content of most of these cells was between 3n and 4n.(ABSTRACT TRUNCATED AT 250 WORDS)