Cyclic mechanical distension regulates renin gene transcription in As4.1 cells

Michael J. Ryan1, Thomas A. Black2, Kenneth W. Gross2, and George Hajduczok1

1 Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo 14214; and 2 Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York 14263


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The renin-producing and -secreting juxtaglomerular (JG) cells are thought to function as the baroreceptor of the kidney. The mechanism by which changes in pressure, or mechanical force, regulate renin at the molecular level has not been elucidated. The renin gene-expressing and -secreting clonal cell line As4.1 was derived from transgene-targeted oncogenesis in mice and was used as a cellular model for JG cells. As4.1 cells subjected to cyclic mechanical distension for a period of 24 h at various frequencies (0.05 or 0.5 Hz) and magnitudes (12 or 24% elongation) were analyzed via Northern analysis for renin mRNA levels. Results indicate that renin gene expression is decreased by 50-85% and returns to basal levels after a 24-h recovery period. Renin gene expression was attenuated independently of elevated cell growth or changes in renin message decay, suggesting that renin gene transcription is directly modulated by mechanical distension. Transient transfection of As4.1 cells with renin 5' flanking sequence-luciferase reporter gene constructs confirmed the role of mechanical stimulation in regulating renin gene transcription. A 43% inhibition of luciferase activity, by stretch, was observed in cells transfected with a 4,000 base pair 5' flanking sequence to the renin proximal promoter. These results demonstrate for the first time that changes in mechanical force can result in the regulation of renin gene transcription and thus provide further insight into the baroreceptor properties of renin-expressing cells.

pressure; expression; transcription; messenger ribonucleic acid stability


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE RENIN-ANGIOTENSIN SYSTEM (RAS) plays an important physiological role in the regulation of arterial blood pressure. Renin, an aspartyl protease, is secreted by juxtaglomerular (JG) cells of the kidney and initiates a biochemical cascade leading to the production of angiotensin II (ANG II). Elevated levels of ANG II lead to arterial vasoconstriction and increased sodium and water reabsorption in the kidney, resulting in an elevated arterial blood pressure. When we consider renin's physiological importance in this cascade, it is not surprising that its production and release are highly controlled.

One of the major constituents regulating renin production and release is blood pressure (8, 16, 29). An inverse relationship between plasma renin activity and renal perfusion pressure has been well documented (16, 22). Presumably, increased vessel wall tension by elevated pressure in the afferent arteriole is responsible for the observed decrease in renin secretion (7). Bock et al. (3) demonstrated the importance of both perfusion pressure and pulsatile pressure on renin release from isolated rabbit glomeruli. The inverse relationship between renin and pressure has led to speculation for the existence of a renal baroreceptor. Blaine et al. (2) first postulated that the JG cells were the baroreceptor on the basis of evidence that the inverse relationship between renin and pressure was still intact in nonfiltering, renal-denervated, adrenalectomized dog kidneys. Moreover, the location of the renin-producing and -secreting JG cell, primarily at the distal portion of the afferent arteriole, places it in a favorable position to sense changes in pressure leading to the glomeruli.

In addition to renin release, renin mRNA levels are also modulated in an inverse fashion by changes in pressure. For example, renin mRNA levels, measured by Northern analysis, were reduced to undetectable levels in rat kidneys hypertrophied in the two-kidney one-clip experimentally induced hypertension model (19). On the other hand, when renal pressure was decreased by abdominal aortic coarctation in the rat, elevated kidney renin mRNA was observed as measured by in situ hybridization. Moreover, the elevated renin mRNA observed in this model was coupled to a recruitment of renin-expressing cells in the region of the afferent arteriole, rather than an elevated production of renin from preexisting JG cells (28).

The inverse relationship between renin and pressure has also been reported in cell culture. Cyclic mechanical stimulation of isolated rat JG cells and the human, renin-expressing, pulmonary tumor cell line CaLu-6 inhibited renin gene expression by 26 and 46%, respectively (6). Although these experiments lend further support to the ability of renin-expressing cells to directly sense and respond to changes in blood pressure, they provide little explanation as to what controls the downregulation of expression. For example, from these experiments, it is not clear whether renin gene expression is reduced as a result of direct transcriptional inhibition or by posttranscriptional modification of the renin message. In addition, it is possible that augmented cell growth by mechanical stimulation could contribute to attenuated renin gene expression such that mitogenic cells might downregulate genes unnecessary for cell division. Cell division, DNA synthesis, and protein synthesis in many cell types, including vascular smooth muscle (13), myocytes (31), endothelial cells (20), and rat mesangial cells (12), are increased by pressure or mechanical force. Whether any, or all, of the aforementioned mechanisms are contributing to the control of renin gene expression by pressure (mechanical force) remains to be determined.

To address these issues, the present study utilizes a renin gene-expressing and -secreting clonal cell line, As4.1, as a model for the JG cell. This cell line was isolated from kidney neoplasm in a transgenic mouse containing a renin SV40 T-antigen transgene that demonstrated appropriate developmental, cell, and tissue-specific expression (27). In addition, As4.1 cells contain large renin-dense granules, making them morphologically similar to bona fide JG cells (15). Previously, our laboratory (K. W. Gross) demonstrated the utility of this model in physiological experiments as 8-bromo-cAMP, appropriately, increased renin secretion (27) and interleukin-1beta downregulated both renin gene expression and transcription (24). Further support for the ability of physiological stimuli to regulate As4.1 cells is provided by our companion study (25). In that study, we demonstrated that mechanical stimulation of As4.1 cells resulted in elevated inositol phosphates mediated by phospholipase C and a rise in intracellular calcium (Ca2+) concentration, which is generally accepted as inhibitory to the release of renin. This evidence provided the rationale for the current study to determine whether mechanical distension of As4.1 cells could, in a physiologically relevant manner, regulate renin gene transcription and expression. Therefore, in these studies, As4.1 cells were subjected to continuous cyclic strain over a 24-h period as a model for pulsatile pressure, with the intention of 1) determining the relationship between renin mRNA levels and mechanical distension, 2) understanding contributions of cell growth and message stability to renin gene control during mechanical stimulation, and 3) elucidating the role of transcriptional regulation of the renin gene by mechanical distension.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. As4.1 cells (ATCC No. CRL-2193) are a renin-expressing clonal cell line derived from the kidney neoplasm of a transgenic mouse (27). Cell cultures were maintained in humidified room air containing 5% CO2 at 37°C. As4.1 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). For RNA analysis, As4.1 cells were cultured 48 h before the experiments onto 6-well Collagen I-coated flexible bottom dishes (Flexcell International) in DMEM with 10% FBS. Twelve hours before the onset of the stretch protocol, cells were placed in serum-free DMEM to synchronize all As4.1 cells in the Go phase of the cell cycle. Two hours before the onset of stretch, cells were placed in DMEM with 10% FBS.

Stretch protocols. As4.1 cells were subjected to stretch of varying frequencies and magnitudes. Renin expression was analyzed after stretch magnitudes of either 12 or 24% membrane elongation at frequencies of 0.05 and 0.5 Hz for a period of 24 h. To determine whether removal of a mechanical stimulus could also regulate renin gene expression, As4.1 cells were mechanically stimulated, as described above, and allowed a 24-h recovery period before RNA analysis. As4.1 cell growth and transfection experiments were performed after mechanical stimulation of 0.05 Hz and 24% membrane elongation for 24 h, and renin message stability experiments were performed over the course of 20 h, also at 0.05 Hz and 24% membrane elongation.

RNA isolation and Northern analysis. Total RNA was isolated from As4.1 cells after mechanical stimulation with the Ultraspec RNA isolation kit (Biotecx). The method was performed according to the manufacturer's instructions and utilizes the guanidium thiocyanate-acid phenol-chloroform approach to RNA purification. Ten-microgram samples of RNA were loaded onto a 2% agarose gel with formaldehyde, so that the RNA could be separated by electrophoresis. Capillary transfer of RNA from the gel to Zeta Probe GT membranes was done overnight in 10× standard sodium citrate, and the RNA sample was permanently fixed to the membrane with short wave ultraviolet illumination. Northern analysis was used to quantify renin mRNA levels with mouse submandibular Ren 2d cDNA as a probe (24). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were unchanged by stretch and were used as an internal control to normalize the renin measurements. Both renin and GAPDH probes were labeled with alpha -[32P]dCTP (Amersham) using a random priming protocol (Pharmacia). The hybridization was performed overnight at 65°C. After incubation with the probes, the blot was washed, and phosphorimagery was used to semiquantify the concentration of renin and GAPDH message.

Renin message stability. Transcription in As4.1 cells was inhibited with actinomycin D (10 µg/ml). Cells were stretched according to the above protocol. At 6, 13, and 20 h after the initiation of stretch, total RNA was harvested from As4.1 cells, and renin mRNA levels were determined using Northern analysis. These results were compared with the level of renin expression at time 0 of the stretch protocol.

As4.1 cell growth. As4.1 cell growth curves were obtained by counting cell nuclei over the course of 3 days. Nuclei were stained with cresyl violet (0.1 g/ml in 0.1% citric acid) and counted with a hemocytometer. Cell nuclei were counted before the onset of stretch, after 24 h of stretch, and after 24 h of stretch with a 24-h recovery period. These results were compared with time-matched control cell counts.

DNA transfections. Luciferase (Luc) derivatives of DNA constructs described previously (26), containing various-length renin upstream regulatory regions, were introduced to As4.1 cells via liposome-mediated transfection by means of Fugene 6 transfection reagent (Boehringer Mannheim). Briefly, Fugene 6 reagent, diluted in Optimem medium, was added to the DNA. This mixture was then added to As4.1 cells in culture on the 6-well stretch plates 2 h before mechanical stimulation. Six microliters of Fugene 6 were used with equimolar amounts of DNA and adjusted to 3.5 µg using Puc19 as carrier DNA. To correct Luc activity for variations in transfection efficiency, As4.1 cells were co-transfected with 200 ng of plasmid containing a Rous sarcoma virus (RSV) promoter driving beta -galactosidase (RSV beta -gal). As4.1 cells were transfected with a promoterless Luc construct (construct a) to determine the background Luc activity.

Luc assay. Immediately after the stretch protocol, medium was aspirated from the 6-well dishes, and cells were washed with phosphate buffer solution (in mM: 137 NaCl, 2.7 KCl, 4.3 Na2HPO4, and 1.4 KH2PO4, pH 7.3). Luc activity (chemiluminescence) was measured using the Luciferase Reporter 1000 Assay System (Promega) in accordance with the manufacturer's instructions. Briefly, 1× reporter lysis buffer was added to the cells to remove them from the cell culture dishes. The cells were collected in microcentrifuge tubes and subjected to a freeze-thaw cycle with dry ice and ethanol to lyse the cells completely. The lysed cells were then pelleted, and the supernatant Luc activity was measured with a Monolight 2010 luminometer (Analytical Luminescence Laboratory). Results are expressed as the percentage of Luc activity of RSV Luc-transfected cells.

beta -Gal assay. beta -Gal activity was measured using Galacto-Light Plus Chemiluminescence Reporter Assay (Tropix) in accordance with the manufacturer's instructions.

Statistical analysis. All data are presented as means ± SE. Data are considered significantly different if P < 0.05, as determined by repeated-measures ANOVA or paired t-tests where noted. Each experiment consists of n = 3 unless otherwise noted.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of cyclic stretch on renin gene expression. As4.1 cells cultured on 6-well flexible bottomed dishes were subjected to cyclic stretch of various magnitudes and intensities for a period of 24 h. Northern analysis of renin mRNA was performed for each experiment and analyzed using phosphorimagery. GAPDH expression levels were used to correct for differences in RNA amounts loaded onto the gel. Figure 1A illustrates data from cells that were stretched with 0 (control), 12, or 24% membrane elongation at a frequency of 0.05 Hz. The results are presented as a percentage of control renin mRNA levels, with the unstimulated ratio of renin to GAPDH representing 100% expression. After the stretch regimen, renin message levels were significantly reduced to 27.6 ± 4.0 and 15.0 ± 1.7% of control at 12 and 24% strain, respectively. To investigate whether there was a frequency effect of stretch, the experiments were repeated at a higher frequency of 0.5 Hz. Figure 1B displays results from those experiments, in which 24 h of 12% stretch reduced renin mRNA to 51.2 ± 7.1%. After the 24% stretch regimen, renin message was reduced to 32.5 ± 7.3% of baseline message levels. The lower expression levels were compared statistically with repeated-measures ANOVA to determine whether there was an effect of increasing stimulus intensity (12-24%) or frequency (0.05-0.5 Hz). No significant difference in renin gene expression between the various stimuli was observed, suggesting that there is maximal inhibition in this system.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 1.   Changes in renin gene expression in response to cyclic mechanical stimulation of increasing intensities (12 and 24% membrane elongation) result in reduced levels of renin expression at frequencies of 0.05 Hz (A) and 0.5 Hz (B). Samples (10 µg) of RNA were used in Northern analysis for renin. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was measured and used to correct differences in gel loading. Significantly (* P < 0.05) different as assessed by repeated-measures ANOVA (n = 3). Higher levels of stimulus intensity and frequency did not produce further reductions in renin expression.

To investigate whether the downregulation of renin gene expression by cyclic stretch was reversible, mechanical stimulation was removed, and As4.1 cells were allowed to recover. These experiments were performed simultaneously with the experiments described above; therefore, results were compared with control and mechanically stimulated levels of renin gene expression. Cells were subjected to a stretch regimen (12% membrane elongation at either 0.05 or 0.5 Hz for 24 h) that preceded a 24-h recovery term (no mechanical stimulation). After this time, renin mRNA levels were measured. Figure 2, A and B, represents results from these experiments. Subsequent to the recovery period, renin message levels were increased to 100.7 ± 2.5 and 121.6 ± 5.6% of control at 0.05 Hz (Fig. 2A) and 0.5 Hz (Fig. 2B), respectively. These values were not statistically different from baseline message levels. Renin mRNA recovery experiments performed with 24% membrane elongation produced similar results (data not shown).


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 2.   Reversal of the inhibition of renin mRNA concentration on abatement of mechanical stimulus [12% membrane elongation at 0.05 Hz (A) and at 0.5 Hz (B)] for 24 h. Samples (10 µg) of As4.1 cell RNA were analyzed by Northern analysis, and variations in gel loading were corrected by measuring GAPDH mRNA. * Statistically different from control (P < 0.001) as evaluated by repeated-measures ANOVA (n = 3).

Effect of cyclic stretch on As4.1 cell growth. Because of the potential for mechanical stimulation to increase cell growth and potentially contribute to the downregulation of renin mRNA, it was necessary to determine whether cyclic stretch augmented As4.1 cell growth. The growth pattern of As4.1 cells was measured over the course of 3 days in culture. Nuclear counts were performed with a hemocytometer in cells stained with cresyl violet. Figure 3 (solid line) represents the growth curve of (mechanically) nonstimulated As4.1 cells. After 24 h, cell number was elevated to 1.8 × 105 ± 9.6 × 103 from an initial seeding density of 8.1 × 104 ± 7.9 × 103 cells. After a 3rd day in culture there was no significant change in cell number. The bottom trace of Fig. 3 depicts the growth curve of As4.1 cells subjected to a stretch regimen (0.05 Hz, 24% membrane elongation) for 24 h (day 2) followed by a recovery that spanned 24 h (day 3). Subsequent to mechanical stimulation, As4.1 cell growth rate was drastically reduced, because there was no significant change in cell number (1.1 × 105 ± 9.5 × 103) compared with control (8.1 × 104 ± 7.9 × 103) cell counts. Furthermore, the reduced number of cells mediated by stretch was significantly lower than its time-matched nonstimulated counterpart. Upon removal of the stretch stimuli, growth rate recovered, and cell number increased until it equaled its time-matched control point during the next 24 h.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 3.   Change in cell growth rate in As4.1 cells subjected to cyclic mechanical strain. As4.1 cell growth was attenuated after cyclic strain (broken line) (24% membrane elongation, 0.05 Hz) compared with nonstretched cells (solid line) over the course of 24 h (day 1 to day 2). Removal of stretch allowed cell growth rate to recover (day 2 to day 3). * Significantly different from each other at time-matched points (P < 0.05 by repeated-measures ANOVA, n = 6).

Effect of cyclic stretch on renin mRNA decay. Actinomycin D (10 µg/ml) was used to inhibit gene transcription in As4.1 cells. This concentration was based on previous studies in our laboratory (K. W. Gross) indicating that 5 µg/ml of actinomycin D inhibited transcription by 95% in As4.1 cells after 1 h (24). Renin message was quantified after 6, 13, and 20 h of mechanical stimulation (0.05 Hz, 24% membrane elongation) and was compared with time-matched controls (nonstretched cells). The half-life of the renin message was 6.9 ± 0.5 h in nonstimulated cells vs. 8.3 ± 0.5 h in mechanically stimulated cells (Fig. 4). These results indicate that cyclic strain does not increase the rate of renin message decay in As4.1 cells. The decay of GAPDH mRNA was also measured, with no change in half-life being observed (data not shown).


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of mechanical strain (24% membrane elongation, 0.05 Hz) on the rate of renin message decay. Samples (2 µg) of As4.1 cell RNA were evaluated for renin expression with Northern analysis at 6, 13, and 20 h after initiation of stretch (time 0). Experiments were performed in triplicate, and data are presented as densitometric units measured by phosphorimagery. Data points were fit with a single exponential decay function.

Effect of mechanical strain on renin gene transcription. Previous studies from our laboratory (K. W. Gross) identified regions of the renin gene as important for transcriptional control (23). A 4,000-bp 5' flanking sequence (-117 to -4,100) of the renin proximal promoter (+6 to -117), fused to a chloramphenicol acetyltransferase reporter gene, resulted in an increase in transcription by two orders of magnitude in an orientation-independent fashion. Within the 4,000-bp upstream sequence, a minimal, 241-bp (-2,625 to -2,866) enhancer was identified that was capable of increasing transcription of the renin proximal promoter 80-fold. Moreover, electromobility shift assays, or EMSAs, were performed, with oligonucleotides spanning the proximal promoter, and these assays identified cell-specific, DNA-protein interactions at the -60 region. Mutations of this -60 region disrupted DNA-protein interactions and abolished transcriptional activity of the promoter when As4.1 cells were transfected with the 4,000-bp construct. Interestingly, cells transfected with the 241-bp enhancer fused to the mutated promoter still demonstrated transcriptional activity, albeit less than what was observed with an intact -60 region (23). These data are consistent with the presence of a negative regulatory element (NRE) located between the promoter and the 241-bp enhancer (-117 to -2,625) (23). Moreover, others have reported an NRE in the Ren 1d mouse renin gene (1, 14, 21, 30). These experiments provided the rationale for the constructs used in the current study. Luc derivatives of the above DNA constructs containing various-length renin upstream regulatory regions fused to the renin promoter were used in liposome-mediated transfections of As4.1 cells. Transfected cells were subjected to cyclic strain (0.05 Hz, 24% membrane elongation) for 24 h, and Luc reporter gene activity was assayed as a measure of transcriptional activity (Fig. 5). When As4.1 cells were transfected with a promoterless Luc construct (construct a) or with the 123-bp renin promoter-Luc construct (construct b), there was no measurable transcriptional activity. Furthermore, there was no significant difference in the transcriptional activity of construct a compared with construct b and, as expected, cyclic strain exhibited no effect. Cells transfected with the 4,000-bp enhancer (-117 to -4,100) fused to the renin proximal promoter (+6 to -117, construct c) displayed a large induction of transcriptional activity, consistent with previous studies (23). After the stretch regimen, transcriptional activity of this construct was reduced from 41.0 ± 3.5 to 24.8 ± 6.8%. Contrary to these findings, the inhibition of transcription was not observed in As4.1 cells transfected with the 241-bp enhancer fused to the renin proximal promoter (construct d). The results of these experiments demonstrate that mechanical strain is capable of downregulating renin gene expression in a transcription-dependent manner. Interestingly, the current data suggest that this regulation requires sequences in addition to the 241-bp enhancer and proximal promoter, providing further support for the presence of an NRE in the renin gene that may be important for the physiological control of renin gene transcription.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 5.   Regulation of the 4.0-kb 5' flanking region of the Ren 2d promoter (P) by mechanical stimulation (24% membrane elongation, 0.05 Hz, 24 h). A luciferase reporter gene (L) was attached to the promoter, as well as various-sized upstream sequences, and transfected into As4.1 cells. RSV Luc, Rous sarcoma virus luciferase. Cyclic strain inhibited transcription when the 4.1-kb fragment was intact (construct c, C) but not when the 241-bp enhancer (construct d, D) alone was transfected. Constructs a and b (A and B) were used as negative controls. Paired t-tests were used to evaluate statistical differences (* P < 0.05, n = 3).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The production and release of renin from the JG cell, although vital to the control of arterial blood pressure, are regulated physiologically by changes in blood pressure. The concept of a baroreceptor mechanism in the kidney has long been hypothesized, in which a sensor capable of responding to changes in pressure has been proposed to exist at the distal portion of the afferent arteriole at the JG cell (2). However, very little is understood about the physiological responses of these renin-expressing cells to alterations in renal arteriolar pressure or mechanical forces. In the accompanying study, we demonstrated that mechanical stimulation of the renin-expressing As4.1 cell opens mechanically active ion channels, initiates the hydrolysis of membrane phospholipids in a phospholipase C- and Ca2+-dependent manner, and elevates intracellular Ca2+ concentration (25). The present study was directed toward determining whether mechanical stimulation of As4.1 cells could result in a change of renin mRNA levels and determining to what extent, if any, transcriptional regulation were involved in that control. These experiments established 1) an inverse relationship between renin mRNA concentrations and cyclic mechanical strain in the renin-expressing As4.1 cell line, 2) renin gene regulation independent of increased cell growth and renin message decay, and 3) compelling evidence for transcriptional inhibition of the renin gene that occurs independently of previously defined promoter and enhancer elements.

The stretch regimens utilized in these experiments were chosen with the intent of modeling pulsatile stretch that may occur in arterial vessels. Over the course of the cardiac cycle, vascular smooth muscle cells can be distended by as little as 5% and as much as 25% with maximum cardiovascular responses (10). If we consider that JG cells are postulated to be modified vascular smooth muscle cells (5) and are in close proximity to the lumen of the afferent arteriole of the nephron, the level of stretch may be commensurate with what is being observed in vivo. Moreover, Nobiling et al. (22) demonstrated that renin release is reduced not only by increased perfusion pressure but also by the changing amplitude of pulsatile pressures. Although the stretch receptor hypothesis in JG cells has been well documented (7, 11, 16), other investigators have been unable to clearly demonstrate a relationship between vessel stretch in isolated glomeruli and renin release. Nonetheless, the authors still identified the JG cell as the kidney's baroreceptor, considering the possibility that the time course of stretch was too rapid to measure, or that mechanical forces could regulate renin via distension of cells in the proximity of the JG cell (3).

Our findings that cyclic strain reduces renin gene expression in As4.1 cells are consistent with recent evidence showing that similar types of mechanical stress inhibited renin gene expression in the pulmonary tumor cell line CaLu-6 as well as primary cultures of rat JG cells (6). These studies demonstrated that mechanical force could directly modulate both basal and stimulated renin release and expression. However, they did not address whether the reduction in renin gene expression by mechanical force was due to transcriptional or posttranscriptional modifications. Moreover, there are some potential limitations to the cellular models used in those experiments. For example, CaLu-6 cells are not kidney derived and express only very low basal levels of renin. Primary JG cells, on the other hand, are kidney derived. Unfortunately, it is difficult to obtain a pure population of JG cells, and those cells that are renin gene expressing lose their ability to express renin over a period of several days in culture (17). These limitations warrant the use of another model, the As4.1 cell, to complement these studies and to gain further insight into the cellular and molecular control of the renin gene by mechanical force. As4.1 cells provide some advantages for studying the physiological and molecular control of renin in vitro. The As4.1 cell is kidney derived, expresses the endogenous renin gene stably over long periods of cell culture, and is morphologically similar to bona fide JG cells (15, 27). Moreover, these cells have been proven extremely useful for identifying cis-acting sequences that are important for the transcriptional regulation of the renin gene. Recent experiments utilizing the As4.1 cell line identified important transcription regulatory regions of the renin promoter and upstream sequence. These experiments have thus far identified two regions important for high-level gene expression: 1) a 241-bp renin enhancer sequence (coordinates -2,625 to -2,866 relative to the transcription start site) and 2) a proximal promoter element sequence located at approximately -60 (23). From experiments described in Petrovic et al. (23), a model of renin transcriptional regulation has been proposed that postulates an NRE located between -117 and -2,625 of the renin 5' flanking sequence. The existence of NRE(s) is required to explain complex interactions observed between the -60 region of the promoter and the enhancer sequences located further upstream. In effect, the binding of trans-acting factors to the -60 region is required to relieve the negative influences of the sequence(s) between the renin promoter and enhancer, permitting enhancement of transcription from the basal promoter.

Transfection of As4.1 cells with Luc constructs containing the 4-kb upstream sequence and the proximal promoter (construct c) was utilized to determine whether changes in mechanical force could directly regulate transcription (Fig. 5). Luc activity, as a measure of transcription, was reduced by ~42%, whereas renin mRNA expression was reduced by 50-85% when cells were mechanically stimulated. The disparity between these values is likely due to the different experimental techniques used. For example, Northern analysis is measuring levels of the endogenous renin gene, whereas the transfections are measuring transcriptional activity on the basis of the assumption that the transfected renin constructs are regulated similarly to the endogenous renin gene. The decreased transcriptional activity may, therefore, be sufficient to explain the decreased renin expression exhibited in Figs. 1 and 2. Also consistent with this finding is that the kinetics of renin mRNA decay, measured after cell transcription was inhibited with actinomycin D, were unaltered by mechanical stimulation (Fig. 4). With a renin message half-life of 8.3 h, one would predict that, after 24 h, ~14% of the renin message would remain. Thus the 50-85% inhibition of renin gene expression observed with mechanical stimulation is consistent with an attenuation of transcription rather than a complete inhibition.

In addition to demonstrating that cyclic stretch regulates renin gene transcription directly and independently of message decay, we examined the effect of mechanical distension on As4.1 cells transfected with a truncated construct in which the 241-bp enhancer was fused directly to the renin promoter (construct d). Use of the 241-bp renin enhancer in transfection analysis would allow us to address the question of whether the promoter or enhancer was involved in the control of the renin gene transcription by mechanical force in As4.1 cells. Interestingly, transcriptional activity was not altered by stretch in the construct containing the 241-bp enhancer (Fig. 5), suggesting that the downregulation of transcription requires 5' flanking sequences in addition to the renin enhancer and proximal promoter. The data in the current study, demonstrating that removal of flanking sequences to the renin promoter and enhancer is sufficient to abolish the inhibitory effect of mechanical force, are consistent with the presence of an NRE, as has been described by other investigators (1, 14, 21, 23, 30). Stretch-responsive elements and transcription factors that bind to the crucial -60 region of the promoter, or in this proposed negative regulatory region, have yet to be identified. However, our recent findings demonstrating that second messengers are activated by stretch in the As4.1 cell (25) provide new insights into cellular pathways that lead to changes in renin gene transcription. On the basis of the accompanying study, which utilized gadolinium (Gd3+), calcium-free Ringer solution, and the phospholipase C inhibitor U-73122 to attenuate the stretch-induced activation of second messengers, it would have been ideal to perform the current gene expression experiments under similar conditions. However, because mRNA message was being assessed, it was necessary to choose protocols that mechanically stimulated As4.1 cells over the course of 24 h. Removal of calcium from the extracellular bath, or otherwise inhibiting second messengers over this time course, could have detrimental effects on basic cell function. Such technical limitations precluded a similar series of studies from being performed over this long time period. Therefore, it remains unclear as to how these second messenger pathways result in the activation or inhibition of transcription factors regulating renin gene activity; this question is certainly an area for further investigation.

It is interesting to note that As4.1 cell growth was not elevated by mechanical strain (Fig. 3) over the time frame examined. Generally, pressure or mechanical stimulation has been implicated in facilitating DNA synthesis and cell growth, which have been shown to contribute to changes in vascular cell wall morphology in spontaneously hypertensive rats (9). Elevated pressure in the glomerulus has been shown to result in glomerulosclerosis mediated by increased mesangial cell growth (18). Pressure augmentation of renin gene-expressing cell division would not fit with the negative feedback relationship that exists, because elevating the number of renin gene-expressing cells could lead to further increases in renin and blood pressure. Furthermore, proliferative cells might downregulate genes unnecessary for the mitotic process, presenting the possibility that the observations of the current work are merely an artifact of increased As4.1 cell growth. Therefore, it was essential that the effects of mechanical force on As4.1 cell growth be tested. The stimulus magnitude of 24% was chosen because it resulted in the greatest effect on renin message. However, this is not to imply that identical results would be obtained if 12% membrane elongation were used. It is entirely possible that, at a smaller stimulus, renin gene expression could be attenuated with no effect on cell growth. Our finding that cell growth does not occur in As4.1 cells subjected to stretch remains consistent, however, with the suggestion that mechanical forces did not increase cell proliferation in CaLu-6 and primary JG cells (6). The As4.1 cell, which was selected after targeting T antigen into renin gene-expressing cells, has a high proliferative index, making it difficult to assess how well it mimics the JG cell in vivo with regard to cell proliferation. However, the results of these experiments are important, because they indicate that proliferation is not providing a trivial explanation for the inhibition of expression and transcription by mechanical force. It is interesting to note that growth patterns of the renin-expressing cells in general, including CaLu-6, primary JG, and now As4.1 cells, are not consistent with what has been observed in most other cell types. Cell growth is induced by mechanical stimulation in a variety of cells, including rat clavarial bone cells (4), rat vascular smooth muscle (9), glomerular mesangial cells (12), bovine aortic endothelial cells (20), and rat myocytes (31). Although it is difficult to compare the clonal renin gene-expressing cells with other primary cultures, it may ultimately be important to discern cellular and molecular mechanisms stimulated by pressure that could lead to hyperplasia in one cell population but not in another.

It should be emphasized that the present investigation, along with our companion paper in this issue (25), was designed to identify a potential connection between mechanical sensing mechanisms at the plasma membrane and the nuclear regulation of renin gene expression. The activation of mechanically sensitive ion channels, which provide a trigger source of Ca2+ whereby subsequent IP3 generation leads to a calcium-induced calcium release process, may certainly subserve such a nuclear role (25). In addition, this rapidly responding signal transduction mechanism may also be involved in the control of renin secretion. Because pools of previously synthesized renin exist in storage granules (29), the signal transduction pathways regulating expression identified in the present study may affect the release of the stored pool of renin. A mechanical stimulus increasing cellular calcium, leading to an inhibition of granular release, the so-called "calcium paradox" of cellular renin regulation, is entirely plausible. Conversely, removal of this inhibitory mechanical stimulus, a maneuver equivalent to decreasing renal perfusion pressure, would likely serve as a positive stimulus for renin secretion. The kinetics of the control of renin secretion are nearly immediate (29). The pathway described in our studies, where renin gene expression is affected by stretch, may represent a parallel path whereby renin could be inhibited or formed de novo to contribute to the renin stores. In a similar manner, in situations where changes in secretion deplete the cellular pool of preformed renin, expression would ultimately be affected to keep up with the demand to secrete. Exactly how this happens, and how this is communicated to the nucleus, are unknown.

We have shown that cyclic mechanical deformation of the renin-expressing cell line As4.1 results in the modification of renin gene transcription and expression. In addition, the application of or removal of stress was concordant with the regulation of renin under conditions of increasing or decreasing renal perfusion pressure, respectively. Taken together with our companion study (25), we have demonstrated a potentially important signal transduction cascade activated by mechanical force in a renin-expressing clonal cell line, As4.1, and we have shown that renin gene transcription in these cells is modulated by mechanical force. Although the precise link between the activation of this second messenger pathway and the transcriptional regulation remains to be determined, the current articles provide new insight into how the baroreceptor properties of renin gene-expressing cells can ultimately be involved in gene regulation. Further experiments will be required to elucidate the connection between the cellular pathways activated by stretch in As4.1 cells and the transcription factors activated downstream of those pathways that ultimately may interact with the proposed NRE located between the renin enhancer and promoter. Importantly, the current system should provide an assay to more precisely delimit the cis-acting sequences in the renin gene that respond to signaling pathways activated by mechanical force.


    ACKNOWLEDGEMENTS

We extend our sincere gratitude to Dr. Craig Jones and Maureen Adolf for their technical expertise, and to the laboratory of Dr. Fred Sachs for assistance with the Flexcell apparatus. We thank Dr. Curt Sigmund (University of Iowa) for providing the derivative luciferase reporter DNA constructs used for transcription experiments.


    FOOTNOTES

This work was supported in part by National Institutes of Health grants HL-49405 (G. Hajduczok), HL-48459 (K. W. Gross), and CA-16056 (K. W. Gross), and American Heart Association Grant 92-310G (G. Hajduczok). M. J. Ryan was partially supported by a Mark Diamond predoctoral grant.

Current address for M. J. Ryan: 2000 Medical Laboratory, Department of Internal Medicine, University of Iowa, Iowa City, IA 52242.

Address for reprint requests and other correspondence: G. Hajduczok, Assistant Professor of Physiology & Biophysics, 126 Sherman Hall, State Univ. of New York at Buffalo, 3435 Main St., Buffalo, NY 14214 (E-mail: pgygh{at}acsu.buffalo.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 13 December 1999; accepted in final form 22 May 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Barrett, G, Horiuchi M, Paul M, Pratt RE, Nakamura N, and Dzau VJ. Identification of a negative regulatory element involved in tissue-specific expression of mouse renin genes. Proc Natl Acad Sci USA 89: 885-889, 1992[Abstract].

2.   Blaine, EH, Davis JO, and Prewitt RL. Evidence for a renal vascular receptor in control of renin secretion. Am J Physiol 220: 1593-1597, 1971[ISI][Medline].

3.   Bock, HA, Hermle M, Brunner FP, and Thiel G. Pressure dependent modulation of renin release in isolated perfused glomeruli. Kidney Int 41: 275-280, 1992[ISI][Medline].

4.   Brighton, CT, Fisher JR, Jr, Levine SE, Corsetti JR, Reilly T, Landsman AS, Williams JL, and Thibault LE. The biochemical pathway mediating the proliferative response of bone cells to a mechanical stimulus. J Bone Joint Surg Am 78: 1337-1347, 1996[Abstract/Free Full Text].

5.   Cantin, M, Araujo-Nascimento MD, Benchimol S, and Desormeaux Y. Metaplasia of smooth muscle cells into juxtaglomerular cells in the juxtaglomerular apparatus, arteries, and arterioles of the ischemic (endocrine) kidney. An ultrastructural-cytochemical and autoradiographic study. Am J Pathol 87: 581-602, 1977[ISI][Medline].

6.   Carey, RM, McGrath HE, Pentz ES, Gomez RA, and Barrett PQ. Biomechanical coupling in renin-releasing cells. J Clin Invest 100: 1566-1574, 1997[Abstract/Free Full Text].

7.   Churchill, PC. Cellular mechanisms of renin release. Clin Exp Hypertens A 10: 1189-1202, 1988[ISI][Medline].

8.   Davis, JO, and Freeman RH. Mechanisms regulating renin release. Physiol Rev 56: 1-56, 1976[Free Full Text].

9.   Diez, J, Fortuno MA, Zalba G, Etayo JC, Fortuno A, Ravassa S, and Beaumont J. Altered regulation of smooth muscle cell proliferation and apoptosis in small arteries of spontaneously hypertensive rats. Eur Heart J 19: G29-G33, 1998[ISI][Medline].

10.   Frangos, JA. Physical Forces and the Mammalian Cell (1st ed.). San Diego: Academic, 1993, p. 400.

11.   Fray, JC, Lush DJ, and Park CS. Interrelationship of blood flow, juxtaglomerular cells, and hypertension: role of physical equilibrium and Ca. Am J Physiol Regulatory Integrative Comp Physiol 251: R643-R662, 1986[ISI][Medline].

12.   Harris, RC, Haralson MA, and Badr KF. Continuous stretch-relaxation in culture alters rat mesangial cell morphology, growth characteristics, and metabolic activity. Lab Invest 66: 548-554, 1992[ISI][Medline].

13.   Hishikawa, K, Nakaki T, Marumo T, Hayashi M, Suzuki H, Kato R, and Saruta T. Pressure promotes DNA synthesis in rat cultured vascular smooth muscle cells. J Clin Invest 93: 1975-1980, 1994[ISI][Medline].

14.   Horiuchi, M, Pratt RE, Nakamura N, and Dzau VJ. Distinct nuclear proteins competing for an overlapping sequence of cyclic adenosine monophosphate and negative regulatory elements regulate tissue-specific mouse renin gene expression. J Clin Invest 92: 1805-1811, 1993[ISI][Medline].

15.   Jones, CA, Petrovic N, Novak EK, Swank RT, Sigmund CD, and Gross KW. Biosynthesis of renin in mouse kidney tumor As4.1 cells. Eur J Biochem 243: 181-190, 1997[Abstract].

16.   Kurtz, A. Cellular control of renin secretion. Rev Physiol Biochem Pharmacol 113: 1-40, 1989[ISI][Medline].

17.   Kurtz, A, Kaissling B, Busse R, and Baier W. Endothelial cells modulate renin secretion from isolated mouse juxtaglomerular cells. J Clin Invest 88: 1147-1154, 1991[ISI][Medline].

18.   Mertens, PR, Espenkott V, Venjakob B, Heintz B, Handt S, and Sieberth HG. Pressure oscillation regulates human mesangial cell growth and collagen synthesis. Hypertension 32: 945-952, 1998[Abstract/Free Full Text].

19.   Moffett, RB, McGowan RA, and Gross KW. Modulation of kidney renin messenger RNA levels during experimentally induced hypertension. Hypertension 8: 874-882, 1986[Abstract].

20.   Murata, K, Mills I, and Sumpio BE. Protein phosphatase 2A in stretch-induced endothelial cell proliferation. J Cell Biochem 63: 311-319, 1996[ISI][Medline].

21.   Nakamura, N, Burt DW, Paul M, and Dzau VJ. Negative control elements and cAMP responsive sequences in the tissue-specific expression of mouse renin genes. Proc Natl Acad Sci USA 86: 56-59, 1989[Abstract].

22.   Nobiling, R, Munter K, Buhrle CP, and Hackenthal E. Influence of pulsatile perfusion upon renin release from the isolated perfused rat kidney. Pflügers Arch 415: 713-717, 1990[ISI][Medline].

23.   Petrovic, N, Black TA, Fabian JR, Kane C, Jones CA, Loudon JA, Abonia JP, Sigmund CD, and Gross KW. Role of proximal promoter elements in regulation of renin gene transcription. J Biol Chem 271: 22499-22505, 1996[Abstract/Free Full Text].

24.   Petrovic, N, Kane CM, Sigmund CD, and Gross KW. Downregulation of renin gene expression by interleukin-1. Hypertension 30: 230-235, 1997[Abstract/Free Full Text].

25.   Ryan, MJ, Gross KW, and Hajduczok G. Calcium-dependent activation of phospholipase C by mechanical distension in renin-expressing As4.1 cells. Am J Physiol Endocrinol Metab 279: E823-E829, 2000[Abstract/Free Full Text].

26.   Shi, Q, Black TA, Gross KW, and Sigmund CD. Species-specific differences in positive and negative regulatory elements in the renin gene enhancer. Circ Res 85: 479-488, 1999[Abstract/Free Full Text].

27.   Sigmund, CD, Okuyama K, Ingelfinger J, Jones CA, Mullins JJ, Kane C, Kim U, Wu CZ, Kenny L, Rustum Y, and Gross KW. Isolation and characterization of renin-expressing cell lines from transgenic mice containing a renin-promoter viral oncogene fusion construct. J Biol Chem 265: 19916-19922, 1990[Abstract/Free Full Text].

28.   Tufro-McReddie, A, Chevalier RL, Everett AD, and Gomez RA. Decreased perfusion pressure modulates renin and ANG II type 1 receptor gene expression in the rat kidney. Am J Physiol Regulatory Integrative Comp Physiol 264: R696-R702, 1993[Abstract/Free Full Text].

29.   Wagner, C, and Kurtz A. Regulation of renal renin release. Curr Opin Nephrol Hypertens 7: 437-441, 1998[ISI][Medline].

30.   Yamada, T, Horiuchi M, Morishita R, Zhang L, Pratt RE, and Dzau VJ. In vivo identification of a negative regulatory element in the mouse renin gene using direct gene transfer. J Clin Invest 96: 1230-1237, 1995[ISI][Medline].

31.   Yazaki, Y, and Komuro I. Role of protein kinase system in the signal transduction of stretch-mediated myocyte growth. Basic Res Cardiol 87: 11-18, 1992[ISI][Medline].


Am J Physiol Endocrinol Metab 279(4):E830-E837
0193-1849/00 $5.00 Copyright © 2000 the American Physiological Society