1 University of Arkansas for Medical Sciences and Veterans Affairs Geriatrics Research, Education, and Clinical Center, Little Rock, Arkansas 72205; 2 Center for Skeletal Aging, Veterans Affairs Medical Center, and Department of Medicine, Medical College of Pennsylvania School of Medicine, Philadelphia 19104; 3 Departments of Medicine and Geriatrics, Mount Sinai School of Medicine, and Bronx Veterans Affairs Geriatrics Research, Education, and Clinical Center, New York, New York, 10029; 4 Biochemistry Laboratories, University of Pennsylvania Veterinary School, Philadelphia, Pennsylvania 19104; and 5 Department of Medicine, University of Cardiff, Cardiff, United Kingdom
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ABSTRACT |
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Herein we demonstrate that replicative cellular senescence in vitro results in sharply reduced inositol 1,4,5-trisphosphate (IP3) receptor levels, reduced mitogen-evoked IP3 formation and Ca2+ release, and Ca2+ store depletion. Human diploid fibroblasts (HDFs) underwent either 30 mean population doublings [mean population doublings (MPDs) thymidine labeling index (TI) >92% ("young") or between 53 and 58 MPDs (TI < 28%; "senescent")]. We found that the cytosolic Ca2+ release triggered by either ionomycin or by several IP3-generating mitogens, namely bradykinin, thrombin, platelet-derived growth factor (PDGF), and epidermal growth factor (EGF), was attenuated markedly in senescent HDFs. Notably, the triggered cytosolic Ca2+ transients were of a smaller magnitude in senescent HDFs. However, the response latency seen with both PDGF and EGF was greater for senescent cells. Finally, a smaller proportion of senescent HDFs showed oscillations. In parallel, IP3 formation in response to bradykinin or EGF was also attenuated in senescent HDFs. Furthermore, senescent HDFs displayed a sharply diminished Ca2+ release response to intracellularly applied IP3. Finally, to compare IP3 receptor protein levels directly in young and senescent HDFs, their microsomal membranes were probed in Western blots with a highly specific anti-IP3 receptor antiserum, Ab40. A ~260-kDa band corresponding to the IP3 receptor protein was noted; its intensity was reduced by ~50% in senescent cells. Thus, we suggest that reduced IP3 receptor expression, lowered IP3 formation, and Ca2+ release, as well as Ca2+ store depletion, all contribute to the deficient Ca2+ signaling seen in HDFs undergoing replicative senescence.
inositol 1,4,5-trisphosphate; fibroblasts; cytosolic calcium; growth factors
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INTRODUCTION |
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THE CYTOPLASMIC RELEASE OF Ca2+ from intracellular stores is critical for the survival, function and propagation of eukaryotic cells. One mechanism through which Ca2+ is released into the cytosol involves activation of microsomal membrane Ca2+ channels gated by the inositol trisphosphate (IP3) receptor (3). That intracellular Ca2+ release becomes defective during cellular senescence is now well established. Namely, senescent human and murine T lymphocytes (12, 28), fibroblasts and neutrophils from elderly humans (19, 29), and parotid cells isolated from aging mice (16) all display attenuated cytosolic Ca2+ signals in response to hormonal and mitogenic stimulation. Paradoxically, fibroblasts harvested from patients with neurodegenerative diseases, such as Alzheimer's disease, exhibit greater agonist-induced Ca2+ release (10, 26). The elevated cellular Ca2+ is thought to contribute to the characteristic neuronal degeneration (10, 26). Despite the somewhat common occurrence of senescence-associated defects in Ca2+ release, we have little insights into their mechanism.
Pathways of cellular Ca2+ influx are also known to be defective in senescent cells, such as the human diploid fibroblast (HDF) cell line WI-38 (20, 23). Most notably, one of our groups observed markedly suppressed Ca2+ currents in HDFs that were induced into replicative senescence through the forced expression of a Ca2+-binding protein specific for Werner's syndrome (20). Senescent WI-38 HDFs also did not show the usual cell cycle-dependent changes in calmodulin expression (4, 22). The latter remained elevated throughout G1, possibly buffering cytosolic Ca2+ to lower levels. In contrast, however, the expression of a different Ca2+-binding protein, calbindin, becomes low in certain neurodegenerative diseases (15), possibly accounting for elevated cytosolic Ca2+ levels.
In the present study, we have used the HDF in vitro aging model to study the molecular mechanism(s) underlying the deficient cytosolic Ca2+ release seen in senescent cells. HDFs have a finite lifespan, and on passage in vitro, undergo replicative senescence. Although the number of mean population doublings (MPDs) accrued before senescence correlates inversely with the donor's age, this concept has recently been revisited (6). In general, however, cells from a given donor can be made to age in vitro and harvested at defined MPDs for studies into their biological characteristics (11, 24, 31).
The HDF cell culture system has been thoroughly validated as an in vitro model of "replicative senescence" (6-8). Notably, these cells exhibit a limited proliferative lifespan. Although HDFs may be at different stages of proliferation/differentiation initially, they ultimately reach a stage on subculturing where they are no longer able to proliferate in response to mitogenic stimuli; this itself is a hallmark of replicative senescence (6-8). In addition to their failure to proliferate, a host of morphological and physiological changes, including the appearance of several molecular markers, characterize HDF senescence (reviewed in Refs. 5-7).
We demonstrate that as HDFs undergo senescence in vitro, their IP3 receptor content and responsiveness to intracellularly applied IP3 falls dramatically. This decline is associated with an attenuated IP3 production and cytosolic Ca2+ release in response to mitogens. As the IP3 pathway is fundamental to intracellular Ca2+ release, its reduction in an aging cell may underlie the cell's defective function and proliferative potential.
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MATERIALS AND METHODS |
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Materials. The Ca2+-sensitive fluorochromes, indo 1 and indo 1-AM, were purchased from Molecular Probes (Eugene, OR). Bradykinin, thrombin, EGTA, and HEPES were purchased from Sigma Chemical (St. Louis, MO). Recombinant human EGF and platelet-derived growth factor (PDGF; BB homodimer) were purchased from R&D Systems (Minneapolis, MN). Ionomycin was obtained from Calbiochem (San Diego, CA).
Cell culture.
HDFs were derived from the anterior forearm skin of a normal 9- yr-old
female, A25 (11). The cells were cultured in MEM supplemented with
fetal bovine serum (15%, vol/vol) and maintained in humidified 5%
CO2. A25 cells have a replicative lifespan of 58 MPDs.
"Young" HDFs are classified as those having a vigorous growth
curve, being within the first half of their maximum MPD, i.e., accruing
30 MPDs with a 3[H]thymidine-labeling index
(TLI) >92%. "Senescent" HDFs are late-passage cells with a
reduced growth potential positioned within the last 10% of their
replicative lifespan, i.e., having accrued between 53 and 58 MPDs (TLI
28%) (11, 19). Both young and senescent HDFs were subcultured in
four chamber slides. They were harvested while proliferating (day
1) for immunobloting, or on confluence (day 3) for
functional studies. In separate experiments, cells were synchronized in
different phases of the cell cycle by incubation with known agents,
namely rapamycin (for Go), KH-93 (for G1), colcemid (for G2) or methotrexate (for S).
Cellular morphometry and microspectrofluorimetry.
We used an ACAS 570 interactive laser cytometer (Meridian Instruments,
Okemos, MI) for single-cell morphometry and microspectrofluorimetry. First, the outline of up to 60 HDFs was traced into a digitizing tablet, and their spread area was computed (in µm2).
Next, cytosolic Ca2+ measurements were made in single HDFs
by using indo 1, a Ca2+-sensitive fluorochrome (for
details, see Ref. 33). Both young and senescent HDFs were incubated
with 5 µM indo 1/AM at 37°C for 60 min in Hanks' balanced salt
solution (HBSS) containing HEPES (HEPES-HBSS, 10 mM) and BSA
(recrystallized, 0.1% wt/vol). The cells were then washed three times
with HEPES-HBSS, incubated for a further 30 min, and positioned on the
microspectrofluorimeter stage. The cells were subjected to excitation
by a 5-W pulsed argon laser at of 357 nm (range, 351-363 nm),
and the emission was monitored every 30 s in the image-scanning mode at
405 and 485 nm. The intensities of the captured fluorescent images at the two wavelengths, I405 and I485, were then
transformed to yield the ratio, I405/I485.
Membrane isolation, SDS-PAGE, and immunoblot analysis.
For the isolation of endoplasmic reticular membranes (2), old and young
HDFs were homogenized in sucrose-mannitol buffer [(in mM) 20 HEPES, 70 sucrose, 220 mannitol, 2 EDTA, 0.1 phenylmethylsulfanylfluoride, as well as 1.25 µg/ml, each, of
antipain, chymostatin, leupeptin, and pepstatin]. The homogenate
was centrifuged (15,000 g, 20 min) to remove mitochondrial and
nuclear membrane fractions. The supernatant was recentrifuged (10,000 g, 1 h; 4°C) to obtain the endoplasmic reticular membrane
fraction. The latter was suspended in homogenization buffer and
repelleted (100,000 g; 1 h). The final pellet was resuspended in 500 µl sucrose-mannitol buffer (above) and stored at
70°C.
RNase protection assay. Complementary RNA for thrombin receptor and epidermal growth factor receptor (EGFR) were synthesized from their respective linearized plasmids by using an in vitro transcription kit (45004K, PharMingen). [32P]-labeled anti-sense RNA probes were hybridized in excess to target RNA in solution, after which free probe and other single-strand RNA were digested with RNase. The remaining "RNase-protected" probes were purified, resolved on polyacrylamide gels, and quantified by autoradiography. All reaction conditions were performed as recommended by the manufacturer (RiboQuant, PharMingen). For quantitation of autoradiograms, the BioRad image-analysis system was used according to the manusfacturer's instructions (Multi-Analyst/PC, version 1.1, BioRad).
IP3 formation assays.
At confluence, young or senescent HDF monolayers were loaded for 24 h
with [3H]myo-inositol (37 kBq/well) in
24-well cluster dishes in inositol-free DMEM containing FBS (1%
vol/vol). Labeled cells were then washed once with 1 ml/well-Hanks'/HEPES buffer (pH = 7.4), and incubated (37°C, 30 min) in the presence of LiCl (20 mM, 290 µl/well). Bradykinin (5 µg/l) or EGF (800 µg/l) were then added in 10 µl of medium, and
the incubation continued for 12 h (37°C). Incubations were terminated by aspiration of the incubation medium and the addition of
900 µl cold (20°C) methanol/HCl (0.12 M, 1:1,vol/vol).
Cells were left a minimum of 2 h (
20°C) before isolation of
total [3H]inositol phosphates in the
supernatant of the disrupted cell monolayers by anion-exchange
chromatography. Eight-hundred-microliter aliquots of the supernatant
were neutralized by the addition of 135 µl NaOH (0.5 M), 1 ml
Tris · HCI (25 mM, pH = 7.0), and 3.1 ml of distilled
water and applied to columns of Dowex I anion exchange resin (8×,
100-200 mesh, chloride form). [3H]inositol
and [3H]glycerophosphoinositol were removed
with 20 ml of distilled water and 10 ml ammonium formate (25 mM),
respectively. [3H]inositol phosphates were then
eluted with 3 ml HCI (1 M), and the columns were regenerated with 10 ml
HCI (1 M) followed by 20 ml distilled water. Radioactivity was
quantified by scintillation counting in the gel phase (Scintillator
Plus, Packard).
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RESULTS |
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Senescent HDFs had a significantly increased spread area (means ± SE,
2,450 ± 174 µm2) compared with that of young cells
(1,718 ± 167 µm2; Student's paired t-test,
P = 0.002, n = 60 cells/group) (24). Cytosolic
Ca2+ measurements were made in up to 100 young or senescent
HDFs that were either proliferating asynchronously or arrested at a
given cell cycle phase (4, 22). We found cytosolic Ca2+
levels were significantly lower in senescent HDFs compared with young
cells in every cell cycle phase, except during G2, when the
difference was reversed (Table 1). Note
that cytosolic Ca2+ levels followed a virtually overlapping
Gaussian distribution in both young and senescent HDFs (not shown).
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Table 2 shows mean peak cytosolic
Ca2+ levels in young and senescent HDFs in response to 5 µM ionomycin either in the presence or absence of extracellular
Ca2+. In both instances, the mean peak cytosolic
Ca2+ level of young HDFs was significantly higher
(P = 0.005 and 0.001, respectively) than that of senescent
cells. In Ca2+-free, EGTA-containing medium, ionomycin will
release Ca2+ from intracellular stores without permitting
Ca2+ influx. The rate of such release should thus be
proportional to the store-cytosol Ca2+ gradient and,
provided the cytosolic Ca2+ level is constant, to the
fullness of the Ca2+ store. In this situation, the rate of
Ca2+ release should thus reflect the fullness of
Ca2+ stores. Figure 1A shows the best-fit curves
for the effect of ionomycin in Ca2+-free, EGTA-containing
medium for young and senescent HDFs. The rate of Ca2+
release was estimated from these plots assuming a near equal, albeit statistically different, basal cytosolic
Ca2+ level (median ~ 5 nM). Although Ca2+ was
released at a rate () of 4.69 nm/s in young cells, this was reduced
dramatically to 0.67 nm/s in senescent HDFs.
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Finally, we used thapsigargin, a microsomal membrane
Ca2+-ATPase inhibitor. By preventing active store
refilling, thapsigargin is known to deplete intracellular
Ca2+ stores. The magnitude of the transient increases of
cytosolic Ca2+ elicited by thapsigargin is thus indicative
of the fullness of intracellular Ca2+ stores. Figure
1B shows that the magnitude of
thapsigargin-induced Ca2+ release in young cells was
significantly greater than in senescent cells.
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Our results with ionomycin and thapsigargin suggested that the store
content of Ca2+ was low in senescent HDFs. We therefore
chose to deplete specifically the IP3-sensitive stores by
using several mitogenic agonists, namely bradykinin, thrombin, PDGF,
and EGF (5, 14, 29, 32). Application of bradykinin (5 µg/l) in either
the presence or absence of extracellular Ca2+ triggered a
rise in cytosolic Ca2+ in both young and senescent HDFs
(Fig. 2A, Table 2). The mean peak
cytosolic Ca2+ level of young cells was, however,
significantly (P < 0.01) higher than that of senescent HDFs
(Table 2). Furthermore, although cytosolic Ca2+ declined to
basal within 100 s of agonist application in young HDFs, this decay was
much slower in senescent cells (Fig. 2A). In contrast, the
application of thrombin (5 kU/l) to HDFs in Ca2+-free,
EGTA-containing medium resulted in a more moderately enhanced cytosolic
Ca2+ level in young cells, with virtually no increase in
senescent cells (Fig. 2B). Again, this resulted in a
significant (P < 0.001) difference between the mean peak
cytosolic Ca2+ in young and senescent HDFs (Table 2).
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The growth factors PDGF or EGF also triggered cytosolic
Ca2+ signals in both young and senescent HDFs. Figure
3 shows that, in both cell populations,
PDGF (10 µg/l) elicited either a transient rise in cytosolic
Ca2+, an oscillatory response, or a biphasic elevation,
consisting of a sharp initial rise followed by a plateau. The cytosolic
Ca2+ signals triggered by EGF were similar to those
elicited by PDGF, except that 1) a smaller overall number of
HDFs responded to EGF; 2) sustained responses were by far never
observed with EGF; and 3) oscillatory responses were observed
in only 14% of EGF-treated senescent HDFs (Fig.
4).
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Table 3 summarizes key data with PDGF and
EGF. First, with either agonist, the number of responding cells was
somewhat higher for young compared with senescent HDFs. Second,
although oscillatory changes were observed more frequently in young
cells, single responses were more common in senescent HDFs. Third, the
peak signal amplitude was significantly greater in young compared with
senescent HDFs. Finally, response latency, defined as the interval
between agonist application and the first response, was markedly
greater in senescent cells after either agonist. It often took as long
as 10 min after EGF application before cytosolic Ca2+
transients were elicited (Fig. 4).
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We next measured cytosolic Ca2+ levels in response to
IP3. Figure 5 shows the
dramatically reduced cytosolic Ca2+ signal triggered by
intracellularly applied IP3 to senescent HDFs. Note that
the plasma membrane of both groups of cells was permeabilized in an
identical protocol by using 60 mg/l saponin (for 5 min) to allow for
the introduction of IP3 into the cytoplasm. Note also that,
as would be expected, basal cytosolic Ca2+ levels of
permeabilized HDFs are higher (~400 nM) than those of
nonpermeabilized cells (~100 nM). As indicated in the MATERIALS AND METHODS, after saponization and fluorochrome loading, the cells were bathed in HEPES-HBSS (cytosolic Ca2+ = 1.25 mM).
We expect that this would allow for some re-sealing of the membrane,
but because of the partially permeable membrane, intracellular
Ca2+ should equilibrate to a new steady-state level; hence
the high basal cytsosolic Ca2+ levels. During this period,
we also measured dye leakage by monitoring photon counts at
I485. Although the fluorochrome did leak over time, the
signal-to-noise ratio remained stable, hence allowing valid
comparisons. It is also important to note that the overall cytosolic
Ca2+ response to applied IP3 was less marked
than, for example, that due to bradykinin (cf. Fig. 2). This could be
due to several reasons: suboptimal IP3 permeation, altered
Ca2+ stores in permeabilized cells, or different (or
additional) signaling pathways used by bradykinin.
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We next compared the content of the IP3 receptor protein in
young vs. senescent HDFs and in confluent vs. proliferating young cells
by Western blotting (2, 34). A highly specific anti-IP3 receptor antiserum, Ab40, raised to the purified human type
I IP3 receptor protein was used (18). Figure
6A shows a Western blot in which
microsomal membranes isolated from young and senescent cells were
probed with Ab40 (see MATERIALS AND METHODS for
details). A ~260-kDa band corresponding to the IP3
receptor protein was seen; the band obtained with senescent HDF
membranes was ~48 ± 14% less intense than that obtained with young
cells. Figure 6B shows that the IP3 receptor band
in proliferating young HDFs was 42 ± 4% less intense than that in
confluent young HDFs. Taken together, the evidence indicates a marked
reduction in IP3 responsiveness and IP3
receptor content in senescent HDFs.
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Moreover, we compared the production of IP3 in young and
senescent cells in response to bradykinin and EGF. The two agonists were chosen because they use distinct mechanisms for IP3
production. Bradykinin interacts with a G protein-coupled receptor,
resulting in the activation of membrane phospholipase C that then
catalyses IP3 formation. In contrast, EGF uses a tyrosine
kinase receptor that phosphorylates a phospholipase C-isoform.
Figure 7 shows the results obtained after a
12-h incubation protocol with two mitogens. Statistical comparisons
were made between young and senescent cells by using Student's
unpaired t-test with Bonferroni's correction for inequality
for significance values <0.05. Notably, senescent cells displayed a
lower IP3 production with both bradykinin and EGF.
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The possibility exists that membrane receptor number had decreased in
senescent cells compared with young cells. We thus examined quantitatively, using the RNAse protection assay, the expression of the
thrombin and EGF receptor. The band intensity for the
thrombin receptor and EGF receptor was similar in both
young and senescent HDFs, indicating the lack of a significant
difference between receptor expression for the respective mitogens
(Fig. 8).
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DISCUSSION |
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We demonstrate that, compared with young cells, senescent HDFs display larger cell spread areas; lower basal cytosolic Ca2+ levels; reduced IP3 formation in response to mitogens; attenuated cytosolic Ca2+ responses to ionomycin, mitogens and IP3; and lowered IP3 receptor protein. Reduced IP3 receptor levels have also been reported in the aging rat cerebral cortex (17, 21). Nevertheless, these receptors have a ubiquitous distribution and are of fundamental importance in Ca2+ homeostasis. A senescence-associated reduction in their expression may therefore explain, at least in part, the attenuated Ca2+ signals observed in aging cells.
We first compared basal cytosolic Ca2+ levels in young and senescent HDFs. The cells were either proliferating asynchronously or were arrested in a specific cell cycle phase (4). The mean basal cytosolic Ca2+ level of senescent HDFs was lower than that of young cells in all phases, except during G2, when the difference was reversed. We believe that these changes, albeit small, represent genuine differences because of a strict calibration protocol used for each cell to calculate its absolute cytosolic Ca2+ level, and high power of analysis (because of a high n value). Indeed, senescent-associated decrements in basal cytosolic Ca2+ may be due, in part, to the enhanced Ca2+buffering, resulting possibly from increased cellular calmodulin (4). By implication, therefore, senescent HDFs synchronized in G2 may express less calmodulin, and hence a higher cytosolic Ca2+. Interestingly, the latter finding is consistent with markedly increased basal cellular Ca2+ levels documented recently in aging human erythrocytes (1).
Next, we compared the fullness of intracellular Ca2+ stores in young and senescent HDFs. In the absence of extracellular Ca2+, an ionophore, such as ionomycin, would deplete all intracellular Ca2+ stores and elevate cytosolic Ca2+ transiently. In this instance, the rate of Ca2+ release would depend on the store-cytosol Ca2+ gradient, and hence, should reflect store fullness (in the presence of near-equal cytosolic Ca2+ levels). The rate of Ca2+ release was reduced by about sevenfold in senescent HDFs. Note that a low store Ca2+ content would not normally result from reduced IP3-sensitive Ca2+ release channels. It should arise from poor store refilling due to either a reduced level of, or a defect in, microsomal membrane Ca2+-ATPases. Store content was further examined by using thapsigargin, a microsomal membrane Ca2+-ATPase inhibitor. Thapsigargin, by blocking Ca2+-ATPAse and preventing store refilling, releases Ca2+ from the store into the cytosol. We found that thapsigargin-induced Ca2+ release was greater in young than senescent HDFs, indicating an expected difference in store Ca2+ content.
We further attempted to 1) trigger the release of
Ca2+ specifically from IP3-sensitive stores and
2) determine whether such agonist-stimulated Ca2+
release was impaired in senescent HDFs. Several mitogens, namely, bradykinin, thrombin, PDGF, and EGF, were applied to both young and
senescent HDFs. Receptors for bradykinin and thrombin generate IP3 by coupling to phospholipase C through the classic
GTP-binding protein Gq. In contrast, tyrosine kinase
receptors for PDGF and EGF phosphorylate and activate a specific
phospholipase C isoenzyme (5, 14, 29, 32). Thus activation of either
class of receptor by its agonist, should, through a distinct mechanism,
generate IP3, and trigger Ca2+ release.
Senescent and young HDFs displayed critical differences in their sensitivity to PDGF and EGF. First, the magnitude of the cytosolic Ca2+ signal triggered in senescent cells was attenuated markedly compared with young HDFs. Second, oscillatory cytosolic Ca2+ changes were much less common in senescent HDFs. Note that oscillatory Ca2+ transients result generally from the rapid and recurrent activation of the IP3 receptor (3). Thus attenuated Ca2+ oscillations can, by themselves, suggest an attenuated IP3 production and/or reduced IP3 receptor numbers. Finally, cells treated with either agonist, PDGF, or EGF showed evidence of a response latency. Again, latency is reminiscent of IP3 receptor activation and is a function of both the IP3 formation rate and IP3 receptor number (3). Senescent cells showed much longer latency intervals, often up to 10 min, compared with young HDFs.
We do not believe that the variation in cytosolic Ca2+
responses to EGF and PGDF in young cells represents cellular
heterogeneity. That all cells respond to bradykinin makes cellular
heterogeneity a less likely explanation for the observed variability in
mitogenic responsiveness. The latter is a well-characterized phenomenon for cells, wherein tyrosine kinase receptors trigger Ca2+
release through IP3 generated from phospholipase C. One
possible reason is that the IP3 generation is much slower
than that for receptors using the G protein-phospholipase C pathway
(3). The generated IP3 may therefore not build up to a high
enough concentration to trigger IP3 receptor-gated
Ca2+ release. In the case of senescent cells, this
phenomenon may become exacerbated, presumably because of reduced
IP3 receptor numbers per se.
Next, we tested, directly, whether senescent HDFs displayed 1) lowered mitogen-induced IP3 formation and 2) reduced IP3 receptor content. Senescent cells showed attenuated IP3 responses to both bradykinin and EGF (and marginally reduced basal IP3 levels). We next examined, in complementary experiments, the effect of IP3 on Ca2+ release, as well as on IP3 receptor protein expression. We found that the peak cytosolic Ca2+ change triggered by intracellularly applied IP3 was fourfold lower in senescent HDFs compared with young cells. In parallel, we also found that the intensity of the 260-kDa protein band seen on Western blotting was ~50% lower in senescent HDFs. Note that the antibody, Ab40, used to probe HDF microsomal membranes has been shown, specifically, to recognize the widely distributed type I IP3 receptor (18). These results, when taken together, are remarkably consistent with, and could possibly explain, the attenuation of mitogen-induced Ca2+ release in senescent HDFs.
Finally, we assessed the expression of receptors for two mitogens, EGF and thrombin using the RNAse protection assay. Such a quantitative method, using GAPDH as a standard, failed to show a significant difference in receptor expression, indicating clearly that the defect in signaling was postreceptor.
Thus we provide compelling functional and biochemical evidence for the attenuation of the IP3-Ca2+ activation pathway, including importantly IP3 receptor expression in aging HDFs. We are by no means claiming that deficiency of this pathway is the sole cause of the poor responsiveness of these cells to mitogenic and hormonal stimulation. Indeed, other molecules, such as surface hormone receptors or, indeed, the cyclic AMP generation machinery (13, 31), might undergo alterations with aging. Nevertheless, the IP3-Ca2+ pathway remains a fundamental component of Ca2+ signaling in eukaryotic cells. Its possible attenuation in other aging cells, such as myocytes, neurones, macrophages, neutrophils, and lymphocytes (10, 12, 19, 27, 28), might underlie, at least in part, their reduced sensitivity to hormonal and mitogenic activation.
Numerous questions still remain unanswered. First, we are uncertain as to whether this loss of responsiveness results from, or else underlies, cellular senescence. Second, it remains unclear whether the more global consequences of aging, such as diminished proliferation, are a consequence of an attenuated IP3-Ca2+ pathway. Third, it remains to be determined whether the expression of other Ca2+ release channels, such as the ryanodine receptor, or indeed plasma membrane cation channels, are also reduced in senescent cells. Finally, and perhaps, more important, our results lay down a firm scientific basis for future studies on the regulation of IP3 receptor gene expression, an area that has been explored poorly to date.
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ACKNOWLEDGEMENTS |
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The authors are grateful to E. J. Moerman for culturing and maintaining HDFs. The intellectual support of Profs. Iain MacIntyre, Stavros Manolagas, and Vincent Cristofalo is gratefully acknowledged.
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FOOTNOTES |
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* M.-S. Huang and O. A. Adebanjo contributed equally to this study.
M. Zaidi was supported by National Institute on Aging Grant RO1-AG 14702 and the Department of Veterans Affairs (Merit Review Award).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. Zaidi, Div. of Endocrinology, Annenberg 5, Mt. Sinai School of Medicine, PO Box 1050, 1 Gustave Levy Place, New York, NY 10029.
Received 12 January 1998; accepted in final form 7 December 1999.
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