IP3, IP3 receptor, and cellular senescence

Ming-Shyan Huang1,*, Olugbenga A. Adebanjo2,3,*, Emmanuel Awumey2, Gopa Biswas4, Antoliy Koval2, Bali R. Sodam2, Li Sun2,3, Baljit S. Moonga2,3, Joshua Epstein1, Samuel Goldstein1,dagger , F. Anthony Lai5, David Lipschitz1, and Mone Zaidi2,3

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 lambda  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.

Basal cytosolic Ca2+ levels were recorded for periods up to 2 min, during which the cells were exposed to prewarmed HEPES-HBSS (Ca2+ = 1.25 mM) containing one of several agonists: ionomycin (5 µM), bradykinin (5 µg/l), thrombin (5 kU/l), PDGF (5, 10, or 20 µg/l), or EGF (100, 400, or 800 µg/l). Experiments with ionomycin, bradykinin, and thrombin were repeated by using the same protocol, but in the Ca2+-free medium (i.e., in 1 mM EGTA).

For experiments with IP3, the plasma membrane was permeabilized gently by exposing HDFs to saponin (60 mg/l, wt/vol; Sigma Chemical), for 5 min at 37°C, in "intracellular buffer" containing (in mM) 110 KCl, 10 NaCl, 2 MgCl2, 5 KH2PO4, 20 phosphocreatine, 10 antimycin A, 3 ATP, and 20 HEPES-KOH, as well as 20 kU/l creatine kinase and 10 mg/l oligomycin (pH = 7.2). Note that we used the intracellular buffer because the cells were permeabilized. Note also that this applied protocol was considered optimal after our multiple trials using different incubation times vs. different saponin concentrations. Permeabilized HDFs were then loaded with 5 µM indo 1 (free acid) in intracellular buffer for 10 minutes at 37°C. The cells were next allowed to reseal for 60 min in HEPES-HBSS (Ca2+ = 1.25 mM), during which time dye leakage was monitored in samples of cells. Although, as with other nonpermeabilized cells, dye leakage did indeed occur over the experimental periods, the signal-to-noise ratio (an indicator of dye retention) was found to be within acceptable limits. The latter was assessed by monitoring photon counts at I485. Finally, chambers containing permeabilized HDFs were mounted on the microscope stage and exposed to prewarmed solutions of IP3 (6 µM).

Indo 1 was calibrated by perfusing the chamber with its free acid solutions (0.5 µM) in 140 mM KCl, 10 mM HEPES, 10 mM EGTA, and different concentrations of CaCl2 (0.02, 1, 2, 3, 4, 5, 7, 8, or 9.0 mM, pH = 6.85 ± 0.01). The Kd for Ca2+ and EGTA (20°C, ionic strength 0.1 M, pH = 6.85) is 747 nM. By using the equation calcium ion concentration ([Ca2+]) = (Kd-1 × [free EGTA])/[Ca - EGTA], these solutions corresponded to [Ca2+] values of 1.5, 83.2, 187, 321, 499, 748, 1,700, 2,990, and 4,650 nM, respectively. The ratio I405/I485 was plotted against the [Ca2+] (for details, see Ref. 34). Note that calibrations were performed at the end of each experiment, so that each cell served as a control.

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.

SDS-PAGE was performed by using 8% separating and 3% stacking polyacrylamide gels using a minigel system (BioRad Laboratories, Cambridge, UK) (35). Microsomes prepared from young and senescent HDFs (100 µg protein) were heated for 5 min at 95°C in loading buffer (2% wt/vol SDS, 2% vol/vol beta -mercaptoethanol, 10% vol/vol glycerol, and 50 mg/l bromophenol blue in 0.1 M Tris · HCl buffer, pH = 6.8). Electrophoresis was performed at a constant current (20 mA/gel). The separated proteins were either stained with Coomassie brilliant bue (Sigma Chemical, Poole, UK) or transferred electrophoretically onto polyvinylidene difluoride membranes (Immobilon, Millipore, Milford, MA) at 15°C for 1 h at 400 mA, then at 1,500 mA for 15 h. The transferred membranes were blocked with nonmilk proteins (4% wt/vol), and then incubated overnight at 4°C with the antiserum Ab40 (1:1,000). After washing and a further incubation with a peroxidase-coupled anti-rabbit antibody, the blot was developed with 3'-diaminobenzidine and hydrogen peroxide. The blots were finally quantitated by using the BioRad Multianalyst Program.

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).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 1.   Resting cytosolic Ca2+ levels in young and senescent human diploid fibroblasts either proliferating asynchronously or synchronized in a given phase of the cell cycle

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 (tau ) of 4.69 nm/s in young cells, this was reduced dramatically to 0.67 nm/s in senescent HDFs.

                              
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Table 2.   Peak cytosolic Ca2+ levels in young and senescent human diploid fibroblasts exposed to either ionomycin, bradykinin, or thrombin

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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Fig. 1.   Least squares fit curves for effect of ionomycin (5 µM) in Ca2+-free, EGTA-containing medium on cytosolic Ca2+ (nM) in young (open circle ) and senescent () human diploid fibroblasts (HDFs; A). Correlation coefficients (r) are shown, and calculated Ca2+ release rates (tau ) are 4.69 and 0.67 nm/s for young and senescent HDFs, respectively. B: time course of effect of thapsigargin (3 µM) in Ca2+-free, EGTA-containing medium on mean cytosolic Ca2+ levels (in nM; values are means ± SE; n = 25) in both young (open circle ) and senescent () HDFs. [Ca2+], calcium ion concentration. * P < 0.01 by ANOVA comparing young vs. senescent HDFs at each time point.

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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Fig. 2.   Time course of effect of bradykinin (5 µg/l; top) and thrombin (5 kIU/l; bottom) in Ca2+-free, EGTA-containing medium on mean cytosolic Ca2+ levels (in nM; values are means ± SE ; n = 24-50 cells) in young (open circle ) and senescent () HDFs. See also Table 2. * P < 0.01 by ANOVA for young vs. senescent HDFs at each time point.

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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Fig. 3.   Representative traces showing time course of effect of platelet-derived growth factor (PDGF; 10 µg/l) on cytosolic Ca2+ levels (nM) in young (top) and senescent (bottom) HDFs. Typical responses include single transient increases (left), oscillatory responses (middle), and biphasic elevations in cytosolic Ca2+ (right). See RESULTS for details. Statistics are as described in Table 3.



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Fig. 4.   Representative traces showing time course of effect of epidermal growth factor (EGF; 800 µg/l) on cytosolic Ca2+ levels in young (A and B) and senescent (C and D) HDFs. Typical responses include single transient increases in cytosolic Ca2+ (A and C), and oscillatory responses (B and D). See RESULTS for details. Statistics are as described in Table 3.

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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Table 3.   Comparison of the effects of the mitogens, platelet-derived growth factor, and epidermal growth factor on cytosolic Ca2+ changes by using a variety of parameters

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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Fig. 5.   Time course of effect of intracellularly applied inositol 1,4,5-trisphosphate (IP3; 6 µM) on mean cytosolic Ca2+ levels (in nM; values are means ± SE; n = 50 cells) in young (open circle ) and senescent () HDFs. * P < 0.01 by ANOVA for young vs. senescent HDFs at each time point.

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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Fig. 6.   Western blot showing ~260-kDa band corresponding to type I IP3 receptor protein when microsomal membranes from young (lane 1) or senescent (lane 2; A), or proliferating (lane 1) or confluent (lane 2; B) HDFs were probed with antiserum, Ab40. Densitometry using BioRad Multianalyst software revealed that mean band density (±SD) of senescent and proliferating young cells was, respectively, 48 ± 14 and 42 ± 4% that of confluent young cells (considered as 100%; n = 3 experiments).

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 Cgamma -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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Fig. 7.   [3H]IP3 formation, shown as counts/min, using an inositol phospholipid hydrolysis assay. HDFs, young (open bars) or senescent (filled bars), were incubated with vehicle (VEH), bradykinin (BRAD; 5 µg/l), or epidermal growth factor (EGF; 800 µg/l) for 12 h, as described in MATERIALS AND METHODS. Values are means ± SE (triplicate estimations in 2 identical experiments; n = 6). * P < 0.05 for young vs. senescent HDFs by Student's unpaired t-test with Bonferroni's correction for inequality.

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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Fig. 8.   RNase protection assay showing bands corresponding to thrombin receptor and EGFR receptor (EGFR), when RNA extracted from both young (lane 2) and senescent (lane 3) HDFs were probed with 32P-thrombin receptor and 32P-EGFR. Lane 1, no RNA (control); lane 4, RNA from testicular cells (control). M, mitogen.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Cgamma 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 Cgamma . 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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

* M.-S. Huang and O. A. Adebanjo contributed equally to this study.

dagger Deceased.

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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