Biomedical subjects

P Kastner

Publications and source records attributed to P Kastner.

At least 73 records · Page 4Linked to original sources

PubMed1992

Retinoic acid receptors and binding proteins in human skin.

Nuclear retinoic acid receptors (RAR) are likely to mediate many of the pleiotypic cutaneous actions of retinoids by acting as ligand-dependent enhancer factors. The presence of nuclear RAR in skin was confirmed by identification of a 45-kDa nuclear RA binding activity by fast protein liquid chromatography (FPLC). Analysis of RNA extracted from skin specimens demonstrated expression of RAR-alpha and RAR-gamma transcripts, as well as expression of the homologous low-affinity receptor, RXR-alpha. Both isoforms of RAR-gamma RAR-gamma 1 and RAR-gamma 2 were detectable, with RAR-gamma 1 being the more strongly expressed. FPLC analysis also demonstrated a 15-kDa peak of specific RA binding activity, consistent with the presence of cellular retinoic acid binding protein (CRABP). Of the two known forms of CRABP, CRABP-II was much more strongly expressed than CRABP-I at the level of steady-state mRNA. CRABP-II was also expressed in keratinocytes and fibroblasts in vitro. CRABP-II was up-regulated by agents that induce keratinocyte differentiation, and inhibited by prolonged exposure to high concentrations of RA. In contrast, CRABP-II was consistently induced by RA in dermal, but not in lung fibroblasts. CRABP-I was expressed at low to undetectable levels under all these conditions. The presence of tissue-specific and differentiation-related regulation of CRABP-II suggests that it may be an important regulator of RA action in human skin.

Carrier Proteins↗

PubMed1992

Differential regulation of retinoic acid receptors and binding proteins in human skin.

Many of the pleiotropic effects of retinoids are likely to be mediated by nuclear retinoic acid receptors (RAR) acting as ligand-dependent enhancer factors. However, in previous studies we have been unable to document altered RAR expression at the RNA level in response to retinoic acid (RA) treatment or in psoriatic lesions, conditions characterized by marked alterations in keratinocyte proliferation and differentiation, which are either caused by or responsive to RA. In an attempt to identify other potential regulators of RA responsiveness, we have used RNA blot hybridization to study the expression of the cellular retinoic acid binding proteins (CRABP) CRABP-I and CRABP-II, the RAR-gamma isoforms RAR-gamma 1 and RAR-gamma 2, and the low-affinity RAR homologue RXR in normal, RA-treated, and psoriatic human epidermis. CRABP-II is selectively and markedly induced by RA in adult human skin (J Biol Chem 266:17662-17666, 1991). However, in submerged, serum-free keratinocyte cultures, CRABP-II mRNA could not be induced by RA. Comparisons of intact human skin, submerged keratinocyte cultures, and human skin equivalent cultures indicated that induction of CRABP-II by RA requires epidermal stratification, dermal-epidermal interactions, or both. CRABP-II transcripts were also expressed in heat-separated human dermis at levels similar to those found in epidermal keratome biopsies, whereas CRABP-I transcripts were undetectable in dermal RNA. CRABP-II transcripts were markedly elevated in psoriatic lesions, as they were in RA-treated skin. In contrast, CRABP-I mRNA was undetectable and not increased in psoriatic lesions. Expression of RAR-gamma isoforms and RXR was not detectably altered in either psoriatic lesions or in RA-treated skin. Thus, altered expression of CRABP-II appears more likely to regulate the cutaneous actions of RA than does altered expression of CRABP-I, RXR, or RAR-gamma isoforms. From these and other results, a model for regulation of RA action involving sequestration of RA by CRABP-II is proposed.

Base Sequence↗

PubMed1992

Retinoid receptors and binding proteins.

Retinoids, in particular all-trans retinoic acid (T-RA), are essential for normal development and homeostasis of vertebrates. Although many effects of retinoids, particularly with regard to teratogenicity, have been described in the literature, the mechanisms by which these simple signalling molecules work has only recently begun to be elucidated. We now recognize at least two classes of retinoid-binding proteins and two families of retinoid receptors. The ultimate interpretation of the retinoid signal within a given cell is probably the result of a complex series of interactions between these proteins, yet little is understood concerning the role each member of this signalling pathway plays. It is therefore imperative to dissect the molecular mechanisms which transduce the effects of these ligands, both in vivo and in isolated systems. One approach we are employing is gene targeting of retinoic acid receptors (RARs) and cellular retinoid-binding proteins to generate mice in which one or more of these genes has been functionally inactivated.

Animals↗

PubMed1992

A retinoid acid 'resistant' t(15;17) acute promyelocytic leukemia cell line: isolation, morphological, immunological, and molecular features.

We report the isolation of a maturation-resistant acute promyelocytic leukemia (APL) cell line. This permanent cell line, derived from the same patient as the maturation inducible NB4 cell line, is the first retinoid-resistant cell line with a t(15;17) chromosomal translocation. Morphological, immunocytochemical and molecular features of the maturation responsive (NB4) and unresponsive (NB4-RAr) cells are compared. The isolation of the NB4-RAr cell line occurred through a two-step process requiring the continuous selective pressure of all-trans-retinoic acid. Cells are also resistant to 13-cis-retinoic acid. Karyotypic and Southern-blot analyses show that the two cell lines are similar with respect to the translocation. Northern-blot analyses show that the chimeric fusion transcript PML-RAR alpha and the normal allelic PML and RAR alpha transcripts are similarly expressed in both cell lines. The molecular basis for unresponsiveness to retinoic acid is not known. This resistant cell line offers a cellular model for molecular biology studies on the mechanism of induction of APL cell maturation, as well as a means to elucidate the molecular mechanisms of resistance. It also furnishes a unique tool for designing and/or screening new therapeutic drugs to avoid or relieve retinoid maturation blockage.

Adult↗

PubMed1991

Multiple isoforms of the mouse retinoic acid receptor alpha are generated by alternative splicing and differential induction by retinoic acid.

Together with the previously described mouse retinoic acid receptor alpha-1 (mRAR-alpha 1, formerly mRAR-alpha 0), we have isolated and characterized here a total of seven mRAR-alpha cDNA isoforms (mRAR-alpha 1 to alpha 7). These isoforms are generated from mRAR-alpha primary transcript(s) of a single gene by alternative splicing of at least eight different exons with the exon which encodes the amino acid sequence of their common B region. All of these isoforms differ in their 5'-untranslated regions (5'-UTRs) and, in the case of mRAR-alpha 1 and alpha 2, also in the sequences encoding the N-terminal A region which is known to be important for differential trans-activation by other members of the nuclear receptor superfamily. In addition, the sequences encoding the open reading frames (ORFs) of mRAR-alpha 3 and alpha 4 cDNA isoforms remain open to their very 5' ends, which suggests that these two isoforms may also encode RAR-alpha s with unique A region amino acid sequences. The two predominant isoforms, mRAR-alpha 1 and alpha 2, were found to be differentially expressed in mouse adult and fetal tissues, as well as in P19 and F9 embryonal carcinoma (EC) cell lines. Interestingly, the expression of mRAR-alpha 2, in contrast to that of the mRAR-alpha 1 isoform, was induced by retinoic acid (RA) in EC cells, thus suggesting the presence of two promoters in the 5' region of the mRAR-alpha gene, which differ in their response to RA. The conservation between mouse and human RAR-alpha 1 and alpha 2 cDNA isoform sequences, as seen by cross-hybridization in Southern blots or by DNA sequence analysis, together with their differential patterns of expression, strongly suggests that they perform specific functions during embryogenesis and in the adult.

Amino Acid Sequence↗

PubMed1991

Differentially expressed isoforms of the mouse retinoic acid receptor beta generated by usage of two promoters and alternative splicing.

Using anchored PCR, three different cDNA isoforms of the mouse retinoic acid receptor beta [mRAR-beta 1, mRAR-beta 2 (formerly mRAR-beta 0) and mRAR-beta 3], generated from the same gene by differential promoter usage and alternative splicing, were isolated. These three isoforms encode RAR proteins with different N-terminal A regions and identical B - F regions. The sequence encoding the first 59 amino acids of the mRAR-beta 3 A region is identical with the entire A region of mRAR-beta 1. However, the sequence of mRAR-beta 3 region A differs from that of mRAR-beta 1 by an additional 27 C-terminal amino acids encoded in an 81 nucleotide-long putative exon which is spliced in between the exons encoding the A and B regions of mRAR-beta 1. Both mRAR-beta 1 and beta 3 cDNAs differ entirely from mRAR-beta 2 in their 5'-untranslated (5'-UTR) and A region coding sequences. This N-terminal variability, in a region which was shown to be important for cell-type specific differential target gene trans-activation by other nuclear receptors, suggests that the three mRAR-beta isoforms may be functionally distinct. The conservation of RAR-beta isoform sequences from mouse to human, as seen by cross-hybridization on Southern blots or DNA sequence analysis, as well as their differential patterns of expression in various mouse tissues, corroborates this view. Additionally, the mRNA analysis data suggest that mRAR-beta 2, whose expression predominates in RA-treated embryonal carcinoma (EC) and embryonic stem (ES) cells, may be important during early stages of development. mRAR-beta 1 and beta 3, on the other hand, which are predominantly expressed in fetal and adult brain, may play some specific role in the development of the central nervous system.

Amino Acid Sequence↗

PubMed1991

Transcriptional regulation of retinoic acid receptor beta in retinoic acid-sensitive and -resistant P19 embryocarcinoma cells.

As in other embryocarcinoma (EC) cell lines retinoic acid (RA) rapidly induces expression of the nuclear retinoic acid receptor (RAR) beta in murine P19 EC cells, while RAR alpha is expressed constitutively. In the RA-resistant P19 EC-derived RAC65 cells, however, there is no such induction and an aberrant (smaller) RAR alpha transcript is expressed. RAR gamma 1 is expressed at low levels in both cell lines. To study the regulation of the RAR beta gene and the possible involvement of RAR alpha protein in transcriptional activation of the RAR beta gene we transfected these cells with a construct containing a 1.6 kb promoter fragment of the human RAR beta gene fused to the CAT gene. Upon transient assays in P19 EC cells CAT activity is enhanced rapidly by RA, to more than 100-fold in a concentration-dependent fashion. On the contrary no activity can be observed in the RA-resistant RAC65 cells; however, co-transfection of hRAR alpha, hRAR beta or hRAR gamma 1 restores the RA-dependent induction of CAT activity. These results clearly show that RAR alpha and RAR gamma 1 can transactivate the RAR beta gene; that RAR beta can stimulate its own expression and that resistance to RA in RAC65 cells is probably due to the altered RAR alpha transcript present in these cells.

Animals↗

PubMed1991

Progestin receptors: isoforms and antihormone action.

We present evidence that the two isoforms of A and B of the chicken (cPR) and human progesterone receptor (hPR) originate from two different mRNA populations. One of these encodes the isoforms A which originate by initiation of translation at an in-frame AUG found 127 (cPR) and 165 (hPR) codons downstream of the AUG which gives rise to the isoforms B. Two estrogen-inducible hPR promoters were identified which are responsible for the generation of these two classes of transcripts. Characterization of the cPR promoter suggested the possible existence of cell-type and isoform-specific auto-regulation of cPR transcription and provided evidence that estrogen-induction of cPR expression occurs at a post-transcriptional level. Finally, we demonstrate promoter-specific transcriptional activation by the hPR isoforms A and B, and we discuss the mechanism of action of the anti-progestin RU486.

Animals↗

PubMed1991

Retinoic acid receptor gene expression in human skin.

Human skin exhibits a characteristic, pleiotypic response to topical retinoic acid. In attempting to understand this response at the molecular level, we have used fast protein liquid chromatography (FPLC) and RNA blot hybridization to characterize the expression of the nuclear retinoic acid receptor (RAR) alpha, beta, and gamma genes in adult human epidermis. Size exclusion FPLC of 0.6 M NaCl nuclear extracts prepared from keratome biopsies revealed two peaks of specific [3H] retinoic acid (RA) binding at Mr 45 and 18 kDa, in agreement with the expected sizes of RAR and cellular RA binding protein. Blot hybridization analysis of total RNA extracted from keratome biopsies revealed that RAR-gamma was the predominant RAR species expressed in human epidermis, as RAR-alpha transcripts were detectable only at low levels and RAR-beta transcripts were undetectable. RAR transcripts were not induced by topical treatment with 0.1% RA cream under occlusion for 4 h or 4 d. Moreover, there was no significant difference in RAR-gamma transcript levels in normal and psoriatic epidermis. RAR-gamma transcripts were constitutively expressed not only in cultured human keratinocytes, but also in human dermal and lung fibroblasts. RAR-beta was induced by RA in dermal fibroblasts, but not in keratinocytes. RA induced IL-1 beta transcripts in keratinocytes rapidly (2 to 4 h) and at low concentrations (3 x 10(-10) M), consistent with activation of the IL-1 beta gene via RAR. These results demonstrate constitutive expression of RAR-gamma in human epidermis, and suggest that RAR-gamma is a molecular target of RA action in adult human skin.

Blotting, Northern↗

PubMed1991

[Fusion proteins between PML and alpha-RAR in acute promyelocytic leukemia].

Acute promyelotic leukemia is characterized by a t(15;17) chromosomal translocation which results in fusion products between PML (chromosome 15) and RAR-alpha (chromosome 17). We describe 2 classes of patients which possess different PMLRAR and RARPML fusion transcripts. We then discuss the functional properties of PML and show that PMLRAR can interfere with both PML and RAR-alpha functions.

Humans↗

PubMed1990

Transient expression of human and chicken progesterone receptors does not support alternative translational initiation from a single mRNA as the mechanism generating two receptor isoforms.

Two isoforms (A and B) of the human (hPR) and chicken (cPR) progesterone receptors originate from a single PR gene. cPR form A results from initiation of translation at a downstream ATG codon (ATG2) which in the cPR cDNA-deduced open reading frame is found 128 amino acids C-terminal to and in-frame with the first ATG codon (ATG1) that gives rise to form B. Our recent observation of an abundant cPR mRNA which encodes only form A suggested to us that the two isoforms are translated from different transcripts (Jeltsch, J. M., Turcotte, B., Garnier, J. M., Lerouge, T., Krozowski, Z., Gronemeyer, H., and Chambon, P. (1990) J. Biol. Chem. 265, 3961-3974). This view is, however, at variance with data obtained by transient transfection with expression vectors containing most of the cPR cDNA (downstream of nucleotide +53) since both isoforms were generated in transiently transfected COS cells (Conneely, O. M., Kettelberger, D. M., Tsai, J. J., Schrader, W. T., and O'Malley, B. W. (1989) J. Biol. Chem. 264, 14062-14064). To further support our above conclusion, vectors containing either hPR or cPR cDNAs were introduced into HeLa and COS-1 cells. Only hPR form B originated from a vector containing the entire cDNA (containing nucleotides 1 to approximately 4400), and form A was produced only from a vector expressing hPR transcripts (nucleotides 814 to approximately 4400) lacking ATG1. Vectors expressing the 5'-untranslated and coding region of the cPR mRNA (nucleotides 29-2921) generated only traces of form A in the two cell lines. Similar traces of form A were observed in COS-1 cells transfected with a vector lacking the 5'-untranslated region. Collectively, these results do not support the hypothesis that similar amounts of the two PR isoforms are generated by alternative initiation of translation on a single PR transcript. We discuss data indicating that for hPR and cPR, isoforms A and B are in fact translated from different mRNAs.

Animals↗

PubMed1990

Two distinct estrogen-regulated promoters generate transcripts encoding the two functionally different human progesterone receptor forms A and B.

The human progesterone receptor (hPR) cDNA, synthesized from T47D breast cancer cells, and the hPR gene 5'-flanking region were cloned and sequenced. Comparison of the cDNA-deduced amino acid sequence with other PR homologues demonstrated the modular structure characteristic of nuclear receptors. As in the case of the chicken homologue, there are two hPR forms, A and B, which originate from translational initiation at AUG2 (codon 165) and AUG1, respectively. Northern blot analysis of T47D mRNA using various cDNA derived probes identified two classes of hPR mRNAs, one of which could code for hPR form B, while the other one lacked the 5' region upstream of AUG1. S1 nuclease mapping and primer extension analyses confirmed that the second class of hPR transcripts are initiated between +737 and +842 and thus encode hPR form A, but not form B. By using the hPR gene 5'-flanking sequences as promoter region in chimeric genes, we show that a functional promoter (located between -711 and +31) directs initiation of hPR mRNAs from the authentic start sites located at +1 and +15. Most importantly, initiation of transcription from chimeric genes demonstrated the existence of a second promoter located between +464 and +1105. Transient co-transfection experiments with vectors expressing the human estrogen receptor showed that both promoters were estrogen inducible, although no classical estrogen responsive element was detected in the corresponding sequences. When transiently expressed, the two hPR forms similarly activated transcription from reporter genes containing a single palindromic progestin responsive element (PRE), while form B was more efficient at activating the PRE of the mouse mammary tumor virus long terminal repeat. Transcription from the ovalbumin promoter, however, was induced by hPR form A, but not by form B.

Amino Acid Sequence↗

PubMed1990

Murine isoforms of retinoic acid receptor gamma with specific patterns of expression.

We have characterized seven murine retinoic acid receptor gamma cDNA isoforms (mRAR-gamma 1 to -gamma 7) generated by alternative splicing of at least seven exons. These isoforms differ from one another in their 5' untranslated region and in two cases (mRAR-gamma 1 and -gamma 2) differ in their N-terminal A region, which is known to be important for differential transactivation by other nuclear receptors. mRAR-gamma 1 and -gamma 2, the predominant isoforms, are differentially expressed in adult tissues and during embryogenesis. Most notably, skin contains almost exclusively mRAR-gamma 1 transcripts. The conservation of the RAR-gamma isoforms from mouse to human together with their patterns of expression suggests that they perform specific functions, which may account for the pleiotropic effect of retinoic acid in embryogenesis and development.

Animals↗

PubMed1989

Differential expression of genes encoding alpha, beta and gamma retinoic acid receptors and CRABP in the developing limbs of the mouse.

Retinoic acid has profound effects on vertebrate limb morphogenesis (refs 1-6, reviewed in refs 7-9), including in the mouse, where it can act as a teratogen generating phocomelia and bone defects. A retinoic acid gradient, possibly amplified by a graded distribution of cellular retinoic acid-binding protein (CRABP), could provide positional information across the antero-posterior axis of the chick limb bud. The discovery of nuclear retinoic acid receptors (RARs) acting as retinoic acid-inducible enhancer factors provided a basis for understanding how retinoic acid signals could be transduced at the level of gene expression. We have now used in situ hybridization to study the distribution of messenger RNA transcripts of the three murine receptors (mRARs) and CRABP during mouse limb development. Both mRAR alpha and mRAR gamma transcripts, but not those for mRAR beta, are present and uniformly distributed in the limb bud at day 10 post-coitum, whereas CRABP transcripts have a graded proximo-distal distribution, indicating that differential expression of CRABP, but not of mRAR alpha or mRAR gamma, could participate in the establishment of the morphogenetic field. At later stages, mRAR gamma transcripts become specific to the cartilage cell lineage and to the differentiating skin and mRAR beta transcripts are mostly restricted to the interdigital mesenchyme. CRABP transcripts, however, are excluded from regions expressing mRAR gamma and mRAR beta. These results indicate that all three RARs and CRABP have specific functions during morphogenesis and differentiation of the mouse limb.

Animals↗

PubMed1989

Cloning of murine alpha and beta retinoic acid receptors and a novel receptor gamma predominantly expressed in skin.

In addition to having profound effects on embryonic pattern formation, retinoic acid (RA) has striking effects on differentiation and maintenance of epithelial cells in vivo and in vitro Skin is a major target organ for retinoids both in its normal and pathological states. The discovery of two human nuclear receptors for RA (hRAR alpha and hRAR beta) acting as transcriptional RA-inducible enhancer factors has provided a basis for understanding how RA controls gene expression. To investigate the specific role that RARs might play during development and in adult tissues, we have cloned the mouse RAR alpha and RAR beta (mRAR alpha and mRAR beta). Their amino-acid sequences are much more homologous to those of hRAR alpha and hRAR beta, respectively, than to each other, which suggests strongly that RAR alpha- and beta-subtypes have different functions. Most interestingly we have discovered a novel RAR subtype (mRAR gamma) whose expression in adult mouse seems to be highly restricted to skin, whereas RAR alpha and RAR beta are expressed in a variety of adult tissues. Furthermore, both mRAR alpha and mRAR gamma RNAs are readily detected in undifferentiated F9 embryocarcinoma (EC) cells, whereas mRAR beta messenger RNA is induced at least 30-fold in RA-differentiated F9 cells.

Amino Acid Sequence↗

PubMed1989

Regulation of progesterone receptor mRNA by oestradiol and antioestrogens in breast cancer cell lines.

The induction of progesterone receptor mRNA by oestradiol and antioestrogens has been characterised in the MCF-7 breast cancer cell line. Progesterone receptor mRNA was induced more than 100-fold by oestradiol. The induction was half-maximal in the presence of 10(-10) M oestradiol and maximum levels were reached after 24 h treatment. Progesterone receptor mRNA was induced to 10% of the oestrogen-induced level by tamoxifen and its metabolite 4'-hydroxytamoxifen. The increase was half-maximal in the presence of 5 X 10(-8) M tamoxifen or 5 X 10(-10) M 4'-hydroxytamoxifen. In contrast, neither the benzothiophene antioestrogen LY117018 nor the 7 alpha-alkyl steroidal antioestrogen ICI 164,384 had any effect on progesterone receptor mRNA. The progesterone receptor mRNA was also induced by oestrogen in a T47D subline and in two other oestrogen-responsive breast cancer cell lines (ZR-75, EFM-19). Tamoxifen was a partial oestrogen for progesterone receptor mRNA induction in each of these cell lines. The large induction of the progesterone receptor mRNA by oestrogen in all 4 breast cancer cell lines supports the contention that the progesterone receptor may be a good predictive marker of hormonal response in human breast cancer.

Animals↗

PubMed1989

A third human retinoic acid receptor, hRAR-gamma.

Retinoic acid receptors (RARs) are retinoic acid (RA)-inducible enhancer factors belonging to the superfamily of steroid/thyroid nuclear receptors. We have previously characterized two human RAR (hRAR-alpha and hRAR-beta) cDNAs and have recently cloned their murine cognates (mRAR-alpha and mRAR-beta) together with a third RAR (mRAR-gamma) whose RNA was detected predominantly in skin, a well-known target for RA. mRAR-gamma cDNA was used here to clone its human counterpart (hRAR-gamma) from a T47D breast cancer cell cDNA library. Using a transient transfection assay in HeLa cells and a reporter gene harboring a synthetic RA responsive element, we demonstrate that hRAR-gamma cDNA indeed encodes a RA-inducible transcriptional trans-activator. Interestingly, comparisons of the amino acid sequences of all six human and mouse RARs indicate that the interspecies conservation of a given member of the RAR subfamily (either alpha, beta, or gamma) is much higher than the conservation of all three receptors within a given species. These observations indicate that RAR-alpha, -beta, and -gamma may perform specific functions. We show also that hRAR-gamma RNA is the predominant RAR RNA species in human skin, which suggests that hRAR-gamma mediates some of the retinoid effects in this tissue.

Amino Acid Sequence↗

PubMed1989

[Microbiologic studies of the lung clearance of rats after aerogenous long term exposure to ammonia].

The effects of aerogenic long-time exposure of 1,990 female Wistar rats to 30, 50, 100 and 150 nl/l of NH3 for 3, 14, 28 and 42 days on clearance of inhaled bacteria (Staphylococcus aureus, SG 511 strain) were studied together with effects on the clinical condition and behaviour of these test animals. For concentrations below 100 nl/l, action upon pulmonary clearance was found to depend above all on the length of exposure, whereas effects of 150 nl/l proved to depend more strongly on concentration. All concentrations tested tended to act on pulmonary clearance in the form of increased numbers of bacteria which remained in the lungs of the experimental animals. Clinical symptoms attributable to NH3 action were not recordable.

Ammonia↗