ARTICLE |
Correspondence to: Isabelle RosinskiChupin, Unité de Biochimie et de Biologie Moléculaire des Insectes, Département Ecosystèmes et Epidémiologie des Maladies Infectieuses, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris, cedex 15, France. E-mail: ichupin@pasteur.fr
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Summary |
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Expression of SMR2, a member of the gene family encoding salivary glutamine/glutamic acid-rich proteins, is regulated by androgens in rat submandibular gland acinar cells. To further characterize SMR2 regulation, we analyzed SMR2 expression during submandibular gland postnatal development and rat puberty at both a global and a single-cell level. Using in situ detection of mature and primary SMR2 transcripts, we show that SMR2 expression is heterogeneous among acinar cells. However, only one cell population with various amounts of mRNAs can be defined. The number of high-expressing cells increases in males during puberty and in females up to 6 weeks of age, suggesting that some factor in addition to acinar differentiation might be important for SMR2 expression in female rats. Involvement of the ß-adrenergic system in regulating SMR2 expression was tested in rats exposed daily to isoproterenol for 4 days. Under these conditions we found an increase in SMR2 expression in female rats, associated with an increase in SMR2 mRNA levels in most acinar cells. This suggests that a signaling cascade, elicited by ß-adrenergic stimuli, might act in concert with androgens to regulate SMR2 expression.
(J Histochem Cytochem 51:13171329, 2003)
Key Words: androgens, ß-adrenergic, salivary glands, glutamic acid/glutamine-rich, proteins, transcription regulation, in situ hybridization
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Introduction |
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DESPITE the enormous advances in understanding transcription regulation, whether or not regulators of transcription serve as binary switches between active and inactive promoter states or as graded regulators of transcription remains a matter of debate. The term "graded" refers to the situation in which a promoter achieves a continuous range of activity from fully on to fully off. Under the "binary" model, the promoter produces only two levels of activity: on or off. Transcriptional activators stimulate expression simply by increasing the likehood that a promoter will be active. In addition to underlying fundamental aspects of gene regulation and cell responses, the occurrence of either binary or graded patterns of responses may be of physiological importance in generating cell subpopulations differing by the nature of the genes they are expressing. Inducers such as steroid hormones are known to elicit typical dose-dependent effects on gene transcription at the level of cell populations. Until recently, patterns consistent with binary responses to inducers were observed in every case in which reporter gene expression was analyzed at the single-cell level (
Two androgen-responsive genes, VCSA1 and SMR2 (
The SMR2 gene is a member of a multigene family encoding salivary glutamine/glutamic acid-rich proteins (GRPs) and proline-rich proteins (PRPs) (
Acinar cells are, along with duct cells, the major cell type of rat SMG, accounting for about 50% of the total cell number. The presence of an androgen receptor in these cells has been demonstrated (
To determine whether the "binary plus graded" model of response to androgens can be generalized to other androgen target genes in the rat SMG, we have studied in more detail SMR2 induction during postnatal development at both a global and a single-cell level. We show that SMR2 expression is dependent on acinar cell differentiation and androgen concentration. As previously observed for VCSA1, SMR2 expression is heterogeneous in the acinar cell population in the glands of female or immature rats. However, in contrast to VCSA1, there is no evidence for a bimodal pattern of SMR2 mRNA level distribution among acinar cells. In addition, SMR2 expression was found to be upregulated after chronic administration of the ß-adrenergic agonist isoproterenol.
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Materials and Methods |
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Animals
Male, female, and pregnant female Wistar rats were purchased from Iffa Credo (Lyon, France). All rats were housed in a temperature-controlled room with free access to food and water. Newborn (no discrimination was made according to the sex of animal) male and female rats of 5, 12, and 18 days or 4, 6, 9, and 11 weeks of age were sacrificed by cervical dislocation or carbon dioxide. For castration experiments, rats were castrated at the age of 4 weeks and sacrificed at 11 weeks. For isoproterenol injection, 8-week-old male and female rats, housed for 1 week, were injected daily with isoproterenol (20 mg/kg body weight; dissolved in 0.15 M NaCl containing 0.3 mM Na2S2O5) or vehicle alone for 4 consecutive days and sacrificed on the next day. In all experiments, care and euthanasia of study animals were in accordance with the European community standards on the care and use of laboratory animals (Ministère de l'Agriculture, France; authorization no. 005329; date 1/26/93). Submandibular glands were dissected and either immediately frozen in liquid nitrogen and stored at -80C before RNA or DNA isolation or were fixed overnight at 4C with 4% paraformaldehyde buffered in PBS.
Probes
The following plasmids were used to generate probes: (a) SMR2 probes: pcS2-1, a 400-bp BglII fragment (nt 132531; GenBank accession number
J05490) cloned in pcDNAII vector (Invitrogen; Leek, The Netherlands) (
RNA Blotting Analysis
RNA was prepared from individual submandibular glands as previously described (
To normalize the results obtained from different Northern blotting experiments and correct for differences of exposure times or transfer efficiency, the same three RNA preparations were included in each experiment and were used to calculate a correction factor. To control the mRNA quality, the blots were hybridized with a ß-actin cDNA probe. Although a decrease in ß-actin mRNA relative quantities in relation to age could be noted during the first weeks after birth (Fig 1A), no significant difference in actin mRNA quantities among individuals at the same age, whatever their sex, were observable, indicating that no sample degradation had occurred.
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Deadenylation Experiments
Deadenylation experiments were adapted from
In Situ Hybridization
In situ hybridization experiments on SMG sections were performed as previously described, with digoxigenin- or 33P- or 35S-labeled cRNA probes (
The proportion of SMG cells expressing SMR2 mRNA at a given age was determined on an average of 5001000 acinar cells by microscopic observation. To count the positive acinar cells on highly labeled sections, we performed the observation under high magnification. An acinar cell was considered as positive when we found a localized higher density of labeling (at least 3 times the background level appreciated on negative cells, e.g., duct cells), associated with a nucleus (for determination of the number of cells, an acinar cell was counted only if its nucleus was detected). When the proportions of labeled cells were very low, the number of acinar cells in a field was estimated and 50150 fields were observed.
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Results |
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Expression of the SMR2 Gene During Post-Natal Development of Rat SMGs
We have previously shown that SMR2 is expressed in the SMG acinar cells in adult male rats (
SMR2 mRNAs are not detected at birth and are barely detected in 5-day-old rats. SMR2 mRNA levels increase thereafter. In contrast to GRP, the plateau is reached only by 6 weeks of age in female rats, 2 weeks after the end of the acinar differentiation period. Differences in SMR2 expression between males and females are apparent as early as 18 days and become statistically significant (p<0.05, Student's test) by 4 weeks of age. In males the plateau is reached by 9 weeks of age. The values for SMR2 mRNA steady-state levels are on average 20-fold higher in males than in females at 11 weeks of age (oscillating between 10- and 100-fold due to individual variations in females).
Altogether, these results are consistent with the fact that the increase in SMR2 expression depends on both acinar differentiation and plasma testosterone concentration. More surprising is the increase in SMR2 expression in females after the end of the acinar differentiation period. This suggests that SMR2 expression might occur at a late stage of acinar differentiation or that it requires, in addition to cell-specific factors, an inducing signal, whose intensity would be maximal by 6 weeks of age in females.
SMR2 mRNAs Are Expressed in Differentiating Acinar Cells
The perinatal development of SMG acinar cells occurs throughout proliferation and differentiation of proacinar or type III cells, which differentiate into seromucous mature acinar cells (
A few GRP mRNA highly positive cells were detected as soon as birth and their number considerably increased by day 5, correlating with the higher level of GRP expression detected by Northern blotting analysis (data not shown). In contrast, only a few SMR2-positive cells were detected at day 5 (Fig 2A). Both the number of SMR2-positive cells and the intensity of labeling were enhanced in 12-day-old rats. However, the proportion of SMR2-positive cells remained low compared with the proportion of GRP-positive cells (Fig 2C). Double hybridization experiments with GRP and SMR2 probes in 5- and 12-day-old rats confirmed that, although most SMR2 highly positive cells also expressed GRP, they represented only a fraction of the GRP-positive cells.
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Using longer (21 days compared to 57 days) exposure times for SMR2 mRNA detection, a large population of acinar cells with labeling intensity near the background level was revealed in addition to the high-expressing cells in 12-day-old rats (not shown). This suggests that, at 12 days of age, all acinar cells might express SMR2 but at very different levels.
To further investigate the differentiation state of SMR2 high-expressing cells, we also performed double ISH experiments at days 5 and 12 after birth with probes for SMR2 and the proacinar marker SMGA (Fig 2B). A number of cells were found to co-express SMR2 and SMGA mRNAs, suggesting that SMR2 high-expressing cells do not necessarily correspond to a late stage in acinar differentiation.
Altogether, these experiments show that SMR2 is likely expressed as early as the type III P stage. In contrast with GRP, whose expression is maximally induced in all differentiating acinar cells, SMR2 is expressed at very heterogeneous levels in differentiating acinar cells.
SMR2 Expression Gradually Increases in Acinar Cells in Males During Puberty and in Females up to 6 Weeks
We next studied SMR2 expression during the first 9 weeks of life in male and female rats, by in situ detection, using medium exposure times (7 days). The percentage of GRP-expressing cells in the entire cell population was determined in parallel to follow acinar cell differentiation. The proportion of GRP-positive cells in the total cell population was found to reach a plateau by 34 weeks of age, suggesting that acinar differentiation might be complete by 4 weeks of age. This is consistent with the fact that, by 4 weeks of age, most acini were found to have gained their typical morphology (
As shown in Fig 3E, the percentage of acinar cells expressing SMR2 increased up to about 6 weeks in females. However, heterogeneity of SMR2 expression levels among acinar cells was conserved (Fig 3A, Fig 3C, and Fig 2D). A population of highly positive cells, which increased in number with age, was observed, together with a large population of cells with medium-intensity labeling. Cells with low SMR2 expression levels, near the background level and not taken into account in the determination of the percentage of positive cells, could be detected at longer exposure times. Therefore, SMR2 expression appears to display a continuous range of values in the acinar cell population in female rats, from low- to high-expressing cells. This is compatible with a continuous distribution of SMR2 mRNA levels around a mean value and an increase in this mean value up to 6 weeks of age in females.
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In males, the number of cells with medium- and high-intensity labeling increased more rapidly than in females and the percentage of these cells among acinar cells reached 100% by 9 weeks of age (Fig 3E). However, some heterogeneity of labeling among acinar cells was maintained up to adulthood (Fig 3B and Fig 3D; see also Fig 4F) and, as in females, the proportion of cells expressing SMR2 at medium levels was high. These results are compatible with a continuous range of SMR2 mRNA levels in the acinar cell population and a progressive increase in the mean SMR2 expression level during puberty.
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A distribution of labeling similar to that in adult females was found in castrated males (not shown).
In conclusion, no evidence for a bimodal pattern of SMR2 expression was obtained under conditions of low or medium concentrations of androgens. Moreover, the continuous increase in the mean SMR2 mRNA levels at the cell level during male puberty strongly suggests a graded induction of SMR2 expression in males. A graded induction of SMR2 expression up to 6 weeks of age is also observed in females.
Detection of SMR2 Primary Transcripts
Increases in SMR2 mRNA steady-state levels, in response to androgens during puberty, might be due to an increase in transcription rate and/or in mRNA half-life. Primary transcripts (hnRNAs), which are temporally associated with the transcription process, are short-lived because of rapid splicing and are therefore considered to be accurate indicators of ongoing or very recent transcription. Therefore, to determine whether the increase in the SMR2 mRNA levels is correlated with a change in transcription rate, we performed in situ detection of SMR2 hnRNAs using an antisense RNA probe corresponding to part of the SMR2 intron 2 sequence. Under these conditions, labeling is observed specifically in the acinar cells, in the SMG of males, before puberty (4-week-old rats), or at adulthood (9-week-old rats), as well as in the SMG of female rats. As expected for primary transcripts, labeling is concentrated in the cell nuclei.
Labeling occurs in a higher percentage of acinar cell nuclei, and is more intense, in adult compared to prepubertal males (40% vs 6%) (compare Fig 4D and Fig 4B), and in males compared to females (40% vs 8%) (Fig 4D and Fig 4C). This indicates that androgen effects on SMR2 expression rely, at least in part, on an increase in transcription rate. Surprisingly, only 4050% of acinar cells contain detectable amounts of hnRNAs in adult 9-week-old male rats. This might be due to the difficulty in detecting low levels of hnRNAs in these experiments. The existence of cells in which SMR2 is transcribed at a lower rate might explain the heterogeneity of SMR2 mRNA levels among acinar cells in male rats and, more generally, in females and immature rats. However, the comparison of SMR2 mRNA and hnRNA labeling patterns on serial sections reveals that some acinar cells contain high amounts of SMR2 mRNA but are negative for hnRNA (Fig 4E and Fig 4F). This suggests that, in these cells, transcription might have recently been turned off, leading to the disappearance of hnRNAs, whereas mRNAs, which are more stable, would still be detected in the cytoplasm. Therefore, SMR2 transcription might be a discontinuous process even in the continuous presence of androgens. The same phenomenon has previously been observed for VCSA1, the second androgen-responsive gene expressed in rat submandibular gland acinar cells (
In contrast, a higher percentage of positive nuclei (Fig 4D and Fig 4G) associated with a lower heterogeneity in mRNA levels among acinar cells is observed using a GRP hnRNA probe or a GRP mRNA probe.
An increase in the percentage of acinar nuclei containing detectable amounts of SMR2 hnRNA is also observed in females between 4 and 9 weeks of age (0.5 vs 8%) (Fig 4A and Fig 4C), correlating with the increase in mRNA steady-state levels and in the number of acinar cells positive for SMR2 mRNA detection.
Repeated Exposure to Isoproterenol Has a Complex Effect On SMR2 mRNA Accumulation, Length of PolyA Tail, and Cellular Distribution
Chronic daily exposure to isoproterenol is known to induce acinar proliferation and differentiation and to affect the expression of a number of acinar genes. For example, isoproterenol has been shown to decrease GRP Ca but not GRP Cb mRNA levels (
To determine if ß-adrenergic agonists could also have an effect on SMR2 mRNA accumulation, we studied the effects of daily IP injection of isoproterenol for 4 days in 8-week-old male and female rats. Northern blotting analysis revealed that repeated administration of isoproterenol increases the accumulation of SMR2 mRNA in both males and females (Fig 5A and Fig 5C), with a slightly stronger effect in females than in males.
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In addition to the increase in amounts of SMR2 mRNA after exposure to isoproterenol, an increase in the size of the hybridizing mRNA was observed in most samples (Fig 5A). To determine the origin of the increase in mRNA size, an increase in the length of the polyA tail was tested. A selective degradation of the polyA tails was performed and the deadenylated mRNAs were analyzed by Northern blotting. As shown in Fig 5B, after deadenylation the SMR2 mRNAs obtained from isoproterenol-injected or control rats migrate with the same apparent electrophoretic mobility, indicating that RNA size differences were due to different polyA tail lengths.
Northern blotting analysis using a GRP probe showed that GRP expression is globally decreased after chronic exposure to isoproterenol (Fig 5A and Fig 5C). Surprisingly, as previously observed for SMR2, the values are slightly more affected in females than in males. In addition, as for SMR2 mRNA, an increase in the mRNA size (Fig 5A) can be observed. This effect also appears to be linked to an increase in the polyA tail length (Fig 5B).
Finally, the effects on SMR2 expression of chronic daily exposure to isoproterenol were studied at the single-cell level by ISH with a probe for SMR2 mRNA. Under these conditions, isoproterenol induces an increase in the volume of acinar cells. In female rats, most acinar cells are now clearly identified as SMR2-expressing cells, consistent with an increase in SMR2 expression per cell. Interestingly, the heterogeneity of SMR2 mRNA levels among acinar cells appears to be reduced, although expression remains higher in a few cells (Fig 6A and Fig 6B). In particular, inside the same acinus all cells express SMR2 at relatively equivalent levels, which contrasts with intra-acinus heterogeneity of labeling in control females, in which high and low expressing cells are found together (Fig 6C). Therefore, chronic daily exposure to isoproterenol has a complex effect on SMR2 expression, not only modifying the levels of SMR2 expression and the length of the polyA tail of the mRNA but also the expression pattern among acinar cells. Despite the increase in SMR2 mRNA levels per cell in females, detection of hnRNA failed to reveal an increase in SMR2 transcription rate in these experiments (data not shown).
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Discussion |
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When studied at the single-cell level, responses to inducers have usually been described as either graded or binary. Using the VCSA1 gene as a model, we previously obtained evidence that, in the physiological context of rat SMG, androgens can elicit both types of responses. To determine whether these androgen effects can be generalized, we have studied the induction of SMR2 expression during postnatal development and during sexual maturation. We show here that the pattern of SMR2 induction differs notably from that observed with either VCSA1 or GRP, a marker of acinar differentiation. In particular, whereas SMR2 is expressed heterogeneously in acinar cells under conditions of low concentrations of circulating androgens (before puberty and in castrated males), the distribution of SMR2 expression levels appears to be unimodal; the androgen response does not include a binary component.
During SMG development, SMR2 expression occurs during the differentiation of acinar cells from their precursors, the so-called type III cells. Several lines of evidence suggest that SMR2, like GRP, is first expressed at the intermediate IIIP cell stage. The first SMR2-expressing cells are detected during the first days after birth, at a time when type IIIP cells are predominant. Furthermore, we found that some cells co-express SMR2 and SMGA, a marker of types III and IIIP cells. However, whereas GRP genes are rapidly expressed at a relatively uniform and high level in most differentiating acinar cells, SMR2 is expressed at very heterogeneous levels in differentiating acinar cells. This heterogeneity is reminiscent of what has previously been observed for VCSA1 (
Beyond this resemblance, there are a number of differences between VCSA1 and SMR2 patterns of expression in acinar cells. We previously demonstrated a bimodal distribution of VCSA1 expression in the acinar cell population in rats before puberty. Two subpopulations that either express VCSA1 at high levels or do not express VCSA1 at all were clearly identified. Although some cells with intermediate labeling levels were observed, they did not constitute a large part of the total cell population. In addition, no further detection of new VCSA1-expressing cells was possible using long exposure times (
Because SMR2 expression depends on acinar differentiation, a surprising observation was that SMR2 mRNA levels continue to increase after the end of acinar differentiation period in female rats and reached a plateau value later than GRP mRNAs. This appears to be specific for SMR2 because VCSA1 expression was previously shown to be maximal in females by 34 weeks of age, like GRP. At the single-cell level, the global increase in SMR2 expression correlated with a gradual enhancement of the SMR2 mRNA content per cell. In addition, a higher number of cells actively transcribing the gene were observed in adult than in 4-week-old females, using in situ detection of primary transcripts, revealing an increase in transcription rate. This suggests that other age-dependent factors, in addition to acinar differentiation, regulate SMR2 expression in females. These factors do not appear to be directly linked to female physiology because both the global levels of SMR2 mRNA and the pattern of SMR2 mRNA distribution were similar in females and in castrated males.
The secretory function of the SMG is regulated, at the level of protein synthesis and secretion, by numerous hormones and by the autonomic nervous system. Both sympathetic and parasympathetic branches of the autonomic nervous system innervate the rat SMG. Whereas parasympathetic innervation of rat SMG is present at birth, sympathetic innervation reaches the glands by postnatal day 5 (
In a search for inducers of SMR2 expression, we looked for an effect of chronic exposure to isoproterenol on SMR2 expression. Isoproterenol was previously shown to be able to induce expression of PRPs, which are phylogenetically related to GRP and SMR2. We show by Northern blotting analysis that chronic injection of isoproterenol induces, particularly in females, an increase in SMR2 mRNA levels while decreasing global GRP mRNA levels. A decrease in GRP-Ca expression was previously reported (
In conclusion, although the precise mechanisms remain to be determined, our results indicate that SMR2 expression can be induced through at least two different pathways, one involving androgens and the other ß-adrenergic signaling. This is reminiscent of previous reports on certain prostatic genes (
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Acknowledgments |
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Supported by grants from the Institut Pasteur and from the Centre National de la Recherche Scientifique.
We would especially like to thank Dr M. Goodhardt for helpful discussions and critical reading of the manuscript.
Received for publication December 23, 2002; accepted May 22, 2003.
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