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Abstract
Objective— Many cell types in atherosclerotic lesions are thought to have various biological abnormalities, such as impaired lipid homeostasis and slow cell proliferation, which may be related to senescence at cellular and individual levels. One of the common characteristics of senescent cells in vitro is the alteration of actin cytoskeletons, which have been reported to be involved in the intracellular transport of lipids. Recently, we raised the hypothesis that Cdc42, which is a member of the Rho-GTPase family and is known to play an important role in actin dynamics, might be important in cellular lipid transport.
Methods and Results— In the present study, we found that the protein expression levels and GTP-binding activities of Cdc42 were decreased in aged human skin fibroblasts. Moreover, we found the intracellular kinetics of Golgi-associated lipids to be retarded in these cells, which was demonstrated by the fluorescence recovery after photobleaching (FRAP) technique and the use of N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-6-aminohexanoyl-d-erythro-sphingosine as a tracer. To correlate the decreased expression of Cdc42 with the retarded FRAP, we complemented the amount of wild-type c-myc–tagged Cdc42Hs (myc-Cdc42Hs-WT) by adenovirus-mediated gene transfer. We further tested the effect of the dominant-active form (myc-Cdc42Hs-DA, V12Cdc42Hs) or dominant-negative form (myc-Cdc42Hs-DN, N17Cdc42Hs) of Cdc42Hs on FRAP. Introduction of myc-Cdc42Hs-WT or myc-Cdc42Hs-DA recovered the retarded FRAP in the aged fibroblasts. Conversely, control fibroblasts infected with myc-Cdc42Hs-DN exhibited significantly retarded FRAP.
Conclusions— These data clearly indicate that the expression of Cdc42, a small G protein, is decreased in the aged cells in close association with the retarded intracellular lipid transport. The present study demonstrates a possible function of Cdc42 in the mediation of intracellular lipid transport.
- aging
- Cdc42
- fluorescence recovery after photobleaching
- intracellular lipid transport
- vesicular transport
Atherosclerotic cardiovascular disease is one of the major causes of death in well-developed countries. The development of atherosclerotic lesions is caused by various cellular dysfunctions as well as modifications of plasma lipoproteins.1 Because the lesions are believed to have an increased local cellular turnover in response to inflammatory changes and because most somatic cells can undergo a finite number of cell divisions before reaching cellular senescence, it had been speculated that many cells in the lesions could have experienced biological aging. It is well known that many cell types, such as macrophages and smooth muscle cells, in atherosclerotic lesions have the following senescence-related characteristics: slow cell proliferation, apoptotic changes, and impaired lipid homeostasis.2–4⇓⇓ These changes are thought to be closely associated with the rupture of cholesterol-rich atherosclerotic plaque, which leads to the onset of acute coronary syndrome, one of the major research foci in basic and clinical cardiology.1
Passaged skin fibroblasts obtained from human subjects have been frequently used as research tools in the fields of lipid and lipoprotein metabolism. It is obvious that the pathophysiology could be elucidated by cell-biological approaches with the use of patients’ fibroblasts in cases of familial hypercholesterolemia and Tangier disease (TD).5,6⇓ Age-related alteration of cellular lipid metabolism has been reported in fibroblasts obtained from aged animals and humans, indicating that lipids such as cholesterol and ceramide accumulate in aged cells.7
One of the major characteristics of cultured aged cells is known to be an enlarged and flattened morphology with altered actin cytoskeletons.8 It has been reported that one of the major determinants for actin cytoskeletons is the Rho-GTPase family,9,10⇓ which is a kind of molecular switch regulating various cell-biological functions with the use of energy produced by the hydrolysis of GTP. From basic science perspectives, an increasing amount of evidence has been accumulated to show that the Rho family has multiple functions that involve not only mediation of the rearrangement of actin cytoskeletons but also the regulation of transcription, adhesion, cell motility, cell cycle, and vesicular transport. However, the pathophysiological and clinical relevance of this family of small G proteins to human diseases has not yet been clarified.11,12⇓
Cdc42, a member of the Rho-GTPase family, was originally identified as a molecule responsible for the budding of yeast as well as the regulation of actin dynamics.9,10⇓ We recently presented the first evidence that the expression of this type of G protein is altered in a human disease by showing that the expression of Cdc42 is reduced in association with the abnormal actin cytoskeletons in cells from patients with TD,13 which is a model for the impairment of intracellular lipid transport and subsequent efflux from the cells.6,14⇓ On the basis of the above data, we have hypothesized that Cdc42 may play a role(s) in cellular lipid transport.
In the present study, we had speculated that some of the Rho-GTPases could be involved in some of the cellular events in senescent cells with an abnormal actin cytoskeletons, and we found the expression of Cdc42Hs to be decreased in aged human fibroblasts. It has been suggested that cytoskeletons such as actins and microtubules could play a role in the vesicular transport of proteins and lipids15,16⇓ and that Cdc42 may play an important role in vesicular transport.9,10,17⇓⇓ Therefore, we sought to determine whether intracellular lipid transport is altered in aged fibroblasts in association with a decreased expression of Cdc42. To analyze intracellular lipid transport, we used fluorescence recovery after photobleaching (FRAP), which is a powerful technique that is used to investigate the intracellular transport of lipids and proteins in living cells.18–20⇓⇓ Using N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-6-aminohexanoyl-d-erythro-sphingosine (C6-NBD-ceramide) as a tracer, we found a retarded intracellular lipid transport in aged living cells. We further proved that this retardation could be related to the dysfunction of the small G protein by using adenovirus-mediated gene transfer of c-myc–tagged wild-type (myc-Cdc42Hs-WT), dominant-active (myc-Cdc42Hs-DA), and dominant-negative (myc-Cdc42Hs-DN) forms of Cdc42. These data would suggest a novel function of Cdc42 in mediating intracellular lipid transport.
Methods
Cells
Eight passaged human skin fibroblast cell lines were used in the present study. Six of them were obtained from Human Science Research Resources Bank (HSRRB, Tokyo, Japan): the donors were 40-, 69-, 72-, 80-, 81-, and 86-year-old women.21 The remaining 2 cell lines (donors were 24- and 48-year-old women) were developed at Osaka University. Informed consent was obtained from all of the donors. For the study of aging, all fibroblast lines were carefully established at HSRRB and Osaka University from normal subjects with no clinical abnormalities. Cells were passaged with a 1:4 split to increase the cumulative cell population doubling level (PDL) by 2 on passage. In the indicated experiments, the 3 cell lines from the 24-, 40-, and 48-year-old women served as controls. The cell lines from women aged >80 years (80-, 81-, and 86-year-old women) served as the aged cell lines. The cells were cultured according to the standard conditions and subjected to the experiments at the indicated PDLs.
Antibodies
Mouse monoclonal anti–c-myc antibody (9E10) and rabbit polyclonal anti-Cdc42 antibody were purchased from Santa Cruz.
Immunofluorescent Microscopy
Immunocytochemical analyses were performed as described previously.13,22⇓ The cells were washed, permeabilized, and then blocked with PBS containing 10% FCS. Primary antibody (anti–c-myc antibody, 1:100 dilution) was applied for 1 hour. After the cells were washed twice, rhodamine-phalloidin (Molecular Probes) and Alexa-conjugated anti-mouse IgG (Molecular Probes) were added to visualize F-actin– and c-myc–tagged Cdc42, respectively. Images were acquired for each fluorescence probe by confocal laser microscopy (LSM 510, Carl Zeiss).
Western Blot Analysis
Fibroblasts were lysed and passed 10 times through 25-gauge needles. SDS-PAGE and Western blot were performed as described previously.13,22⇓
Cdc42 Activation Assay
GTP-bound Cdc42 was assessed by a Cdc42 Activation Assay Kit (Upstate Biotechnology) according to the manufacturer’s protocol.23 Briefly, cells were lysed with lysis/wash buffer containing 10 mmol/L MgCl2 (Upstate Biotechnology), and lysates were immediately added to p21-activated kinase-1/p21-binding domain agarose. For a positive control, lysate was incubated in vitro with GTPγS. After the reaction mixture was incubated for 1 hour at 4°C, the beads were washed 3 times and resuspended in Laemmli sample buffer. Samples were separated by SDS-PAGE, followed by Western blot to detect GTP-bound Cdc42.
Construction of Adenovirus Vectors and Their Expression in Fibroblasts
The cDNAs of myc-Cdc42Hs-DA (V12Cdc42Hs) and myc-Cdc42Hs-DN (N17Cdc42Hs) were kindly provided by Dr Kenji Takaishi and Prof Yoshimi Takai (Department of Molecular Biology and Biochemistry, Osaka University, Osaka, Japan).24 The cDNA of myc-Cdc42Hs-WT was cloned by reverse transcription–polymerase chain reaction, as described previously.25 Adenovirus vector encoding myc-Cdc42Hs-DA was constructed by an adenovirus expression kit with the use of the COS-TPC method (Takara).26 Adenovirus vectors encoding LacZ, myc-Cdc42Hs-WT, and myc-Cdc42Hs-DN were constructed according to the protocol of the Adeno-X expression system (Clontech).27 The titer of the virus stock was assessed by a plaque formation assay using HEK293 cells and was expressed as plaque formation units. Infection with adenovirus was carried out by incubating the cells in serum-free medium for 1 hour at 37°C under a gentle agitation. After incubation, complete medium was supplied, and the cells were further incubated in a CO2 incubator. Five days after infection with the indicated multiplicity of infection (MOI), the cells were applied to the experiments.
Fluorescence Recovery After Photobleaching
FRAP experiments were carried out as follows19,20,28⇓⇓: Cells were stained with C6-NBD-ceramide (Molecular Probes)28 for 30 minutes at 4°C. After 2 washings with ice-cold PBS, the cells were incubated with complete medium at 37°C for 30 minutes. A beam of light using 488-nm laser lines was focused on the indicated part of C6-NBD-ceramide–positive regions in the living cells by confocal laser microscopy (LSM 510, Carl Zeiss). Typically, 20 to 25 iterations were required for almost complete photobleaching. After the appropriate bleach pulse, FRAP was monitored at the bleached area until 30 seconds after bleaching. The relative fluorescence was measured by dividing the fluorescence in the FRAP area by that in the reference spot. The recovery was reasonably fit by a single exponential function. After the values of relative fluorescence were plotted, the time constant was calculated.
Results
First, we examined the effect of in vivo and in vitro aging on the expression of immunoreactive mass of Cdc42 in human skin fibroblasts. In Figure 1A, the relationship between the age of donors and the expression levels of Cdc42Hs was plotted. Whole-cell lysates from skin fibroblasts with the same PDL (PDL 24) were subjected to Western blot analyses. It appeared that the expression of Cdc42 declined along with aging (Figure 1A). The statistical analyses confirmed that the expression of Cdc42 was significantly lower in the cell lines from the subjects aged >80 years (n=3; the donors were aged 80, 81, and 86 years) than in the cell lines from control subjects (n=3; the donors were aged 24, 40, and 48 years; P<0.01; Figure 1B). In addition to the protein expression levels, we further investigated the GTP-binding activity of Cdc42. As shown in Figure 1C, the GTP-bound Cdc42 was significantly decreased in the cell lines from the subjects aged >80 years (P<0.01). Online Figure I (please see http://atvb.ahajournals.org) shows the effect of in vitro aging on the expression of immunoreactive mass of Cdc42. The control cell lines (n=3; the donors were 24, 40, and 48 years) were cultured, and the cell lysates were obtained at different PDLs (PDLs 8, 28, and 48). The expression of Cdc42 was significantly decreased in the cells with higher PDLs, suggesting that in vitro aging also induced a reduction of Cdc42.
Figure 1. Expression of Cdc42Hs in human passaged fibroblasts from aged subjects and its changes with in vitro aging. A and B, Expression of immunoreactive mass of Cdc42Hs in human fibroblasts obtained from donors of different ages was examined by Western blot analyses. All cell lines were subjected to this experiment at PDL 24. Rabbit anti-Cdc42Hs polyclonal antibody (1:2000 dilution, Santa Cruz) was used as the primary antibody. The immunoreactive mass in each cell was plotted to show a possible age-related decline of the expression of Cdc42 (A). Statistical analyses were performed between the 3 control cell lines obtained from 24-, 40-, 48-year-old women and the 3 aged cell lines obtained from 80-, 81-, 86-year-old women (B). Probability values were calculated by Student t test. The expression of Cdc42Hs was significantly decreased in the fibroblasts from the subjects aged >80 years (P<0.01). C, GTP-bound Cdc42 was assessed by Cdc42 activation assay kit (Upstate Biotechnology), when all cell lines were at PDL 24. At the top, a Western blot is shown. The lane showing GTPγS denotes the positive control experiment. Probability values were calculated by Student t test. Statistical analyses showed that the immunoreactive masses of GTP-bound Cdc42 in human fibroblasts at PDL 24 were significantly decreased in the cell lines from aged subjects (P<0.01).
Next, we examined intracellular lipid transport in the aged cells by using the FRAP method, which is a powerful technique that is used to investigate the intracellular transport of lipids as well as proteins in living cells.18–20⇓⇓ We have tested the lateral mobility of lipids in the Golgi apparatus by FRAP with the use of a fluorescent ceramide (C6-NBD-ceramide, Molecular Probes) as a tracer. Some previous studies have reported that the kinetics of C6-NBD-ceramide closely reflects that of cholesterol.19,20,28⇓⇓ After incubation, C6-NBD-ceramide accumulated around the Golgi apparatus (please see online Figure IIA through IIC, available at http://atvb.ahajournals.org). After bleaching, the recovery of fluorescence intensity was monitored (Figure IID), and time constants were measured in the defined region. We found that the time constants for recovery were significantly prolonged in the cell lines from the subjects aged >80 years (n=3; the donors were aged 80, 81, and 86 years) compared with those of the control subjects (n=3; the donors were aged 24, 40, and 48 years; 7.5±2.0 versus 12.2±2.2 seconds, respectively [P<0.05]; Figure 2A). The time constants were also significantly prolonged in the cells with higher versus lower PDLs (n=3; the donors were aged 24, 40, and 48 years; 7.5±2.0 versus 15.3±3.0 seconds, respectively [P<0.01]; Figure 2B). We noted that the time constant in cells from a TD patient13,14⇓ was prolonged, which was compatible with the results of a previous report by Orso et al.19 These data indicated that intracellular lipid transport in the Golgi apparatus was retarded in skin fibroblasts from aged human subjects as well as in the cells with in vitro aging.
Figure 2. Retarded intracellular lipid transport in fibroblasts from aged subjects and its changes with in vitro aging. A, Effect of in vivo aging on FRAP. The 3 cell lines from 24-, 40-, and 48-year-old women served as controls. As in vivo aged cell lines, the 3 cell lines from 80-, 81-, and 86-year-old women were analyzed. All cell lines were subjected to the experiments at PDL 24. In each cell line, 10 cells were subjected to FRAP analyses, and time constants were measured. Mean values are expressed as bar graphs, and statistical analyses were performed between control and in vivo aged fibroblasts. Probability values were calculated by Mann-Whitney U test. B, Effect of in vitro aging on FRAP. The control cell lines at different PDLs (PDL 24 and 48) were analyzed by the FRAP technique. Mean values are expressed as bar graphs, and statistical analyses were performed. Probability values were calculated by Mann-Whitney U test.
The above findings gave us a hypothesis that Cdc42 may regulate intracellular lipid transport in the aged fibroblasts. To prove this, we attempted to introduce the following Cdc42 constructs into these cells by using the adenovirus-mediated gene transfer. We made adenovirus vectors encoding the wild type of Cdc42Hs (myc-Cdc42Hs-WT) as well as its dominant-active (myc-Cdc42Hs-DA [V12Cdc42Hs]) and dominant-negative (myc-Cdc42Hs-DN [N17Cdc42Hs]) forms. As shown in online Figure IIIA through IIIC (available at http://atvb.ahajournals.org), when fibroblasts infected with adenoviruses encoding myc-Cdc42Hs-WT, -DA, or -DN were analyzed by Western blot, each construct was overexpressed in an MOI-dependent manner. We confirmed the expression of the transgene by immunocytochemical analyses as well (online Figure IV, available at http://atvb.ahajournals. org). The cells were stained with anti–c-myc antibody and rhodamine-phalloidin to visualize transgene expression (green) and actin cytoskeletons (red), respectively. As shown in online Figure IVA, IVD, and IVF, when the fibroblasts were infected with the recombinant adenovirus with 150 MOI, transgene expression could be detected in all the cells observed. These data indicated that myc-Cdc42-WT, -DA, and -DN were successfully introduced into the fibroblasts by adenovirus-mediated gene transfer. Some morphological changes were observed in fibroblasts infected with adenoviruses encoding myc-Cdc42-WT (Figure IVA through IVC), myc-Cdc42-DA (Figure IVD and IVE), and myc-Cdc42-DN (Figure IVF and IVG). As shown in Figure IVD and IVE, fibroblasts infected with adenovirus encoding myc-Cdc42-DA exhibited the development of filopodia formation, which was consistent with previous reports.9,10⇓ As shown in Figure IVA and IVF, fibroblasts infected with adenovirus encoding myc-Cdc42-WT or -DN did not exhibit manifest morphological changes. However, we could observe that few cells strongly expressing myc-Cdc42-WT exhibited quite similar morphology with cells expressing myc-Cdc42-DA (Figure IVC).
Finally, to elucidate the contribution of the decreased expression of Cdc42Hs to retarded intracellular lipid transport, we performed a FRAP analysis in the aged fibroblasts infected with the adenoviruses. Online Figure VA and VB (available at http://atvb.ahajournals.org) shows the representative images of FRAP in aged and control cell lines infected with the adenovirus constructs, respectively. The recovery of fluorescence was evaluated as time constants, and the results are summarized in Figure 3. As shown in Figure 3, the complementation of the wild-type Cdc42Hs completely corrected the retarded FRAP in the aged cells, whereas we could not observe any differences of FRAP between cells infected with and without adenovirus encoding LacZ. We also analyzed the effect of the introduction of myc-Cdc42-DA and -DN into these cells on intracellular lipid transport. The time constants were also significantly shortened by adenovirus-mediated introduction of myc-Cdc42-DA in the aged cells (8.0±1.7 seconds), whereas infection with myc-Cdc42-DN prolonged the time constants in the aged cells (17.5±2.5 seconds). As shown in Figure 3, FRAP was significantly retarded in control fibroblasts infected with adenovirus encoding myc-Cdc42Hs-DN compared fibroblasts infected with adenovirus encoding LacZ (16.8±2.8 versus 7.3±2.3 seconds, respectively; P<0.01). These results strongly support the hypothesis that Cdc42Hs plays an important role in regulating intracellular lipid transport, suggesting a novel function of Cdc42, a small G protein, in mediating intracellular lipid transport.
Figure 3. Effect of infection of adenoviruses encoding LacZ, Cdc42Hs-WT, Cdc42Hs-DA, and Cdc42Hs-DN (MOI 150) on the time constants of FRAP. The control (n=3; donors were aged 24, 40, and 48 years) and aged (n=3; donors were aged 80, 81, and 86 years) cell lines were infected with the indicated adenoviruses. All cell lines were used at PDL 24. In each cell line, 10 cells were subjected to FRAP analyses. Bar graphs indicate the time constants of FRAP in the control and aged fibroblasts infected with adenoviruses encoding LacZ, Cdc42-WT, Cdc42Hs-DA, and Cdc42Hs-DN. *P<0.05 and **P<0.01 by Mann Whitney U test.
Discussion
In the present study, we found an age-related decline of the expression of Cdc42 in human skin fibroblasts in association with retarded intracellular lipid transport, demonstrated by the FRAP technique. We further found that the GTP-bound Cdc42 was also decreased in the aged cells. As in many previous experimental studies, we examined the effect of dominant-active and dominant-negative mutants of Cdc42 on the phenotype of interest, intracellular lipid transport. The dominant-active form shortened and dominant-negative form retarded the time constants in the fibroblasts tested. In addition, we found that the complementation of wild-type Cdc42Hs successfully corrected the retarded transport. These results strongly support our hypothesis that this small G protein plays an important role in intracellular lipid transport.
The ceramide we used in the present study, C6-NBD-ceramide, is believed to be metabolized and accumulated in the Golgi apparatus and to be sorted and transported to the plasma membrane via vesicular transport.18,19,28⇓⇓ It is also thought that the kinetics of C6-NBD-ceramide in the Golgi apparatus is closely correlated with that of cholesterol for the following reasons: This kind of ceramide is an efficient substrate for glucosylsphingolipid and sphingolipids, both of which are known to be assembled together with cholesterol to form a cholesterol-sphingolipid microdomain or raft.29 The FRAP of the ceramide can be affected by cholesterol deprivation and repletion.28 Collectively, one of the plausible implications from the present data is that the altered lateral mobility observed could be related to the impairment of intracellular transport and the subsequent export of cholesterol from the cells.
It has been reported that cells obtained from TD patients with ATP-binding cassette transporter-1 (ABCA1) abnormalities exhibit significantly retarded lateral mobility of C6-NBD-ceramide.19 In the present study, we obtained similar results in our TD cells (Figures 2A and 3⇑). Recently, it has been demonstrated that ABCA1 itself moves between intracellular compartments via vesicular transport, suggesting that this molecule could be involved in intracellular lipid transport as well as the exporting of lipids.30 We have previously reported that the expression of Cdc42 is decreased in cells from TD patients and that the introduction of myc-Cdc42Hs-DN decreases cholesterol efflux from the transfected cells.13 We have recently demonstrated that ABCA1 and Cdc42 could colocalize and have a possible protein-protein interaction in the transfected cells.25 These 2 molecules and others might synergistically work and contribute to the intracellular lipid transport as well as the exporting of lipids from the cells.
It would be of importance to know the mechanism for the decreased expression of Cdc42Hs as well as the reduction of GTP-bound Cdc42 in the aged cells. Because our preliminary data showed that Cdc42 mRNA appears to be decreased in those cells, the reduction in immunoreactive mass of Cdc42 and GTP-bound Cdc42 could be explained at least in part by some alteration at mRNA levels. In addition, we reported that the expression levels of Cdc42 mRNA and protein were decreased in cells from patients with TD. Although we could consider some possibilities, such as the involvement of telomerase, further studies are required to elucidate the molecular mechanism.
Cdc42 is a member of the Rho-GTPase family (from the Ras superfamily of monomeric 20- to 30-kDa GTP-binding proteins).9,10⇓ It would be of importance to know the expression levels of other Rho family members, such as Rac I and Rho A, and whether they have some function in the mediation of intracellular lipid transport. It is also important to know what kinds of effectors of Cdc42 are involved in intracellular lipid transport, because the Rho family members are believed to be a kind of molecular switch that activates the downstream effectors. Previous experimental studies have demonstrated that Cdc42 regulates a variety of essential cellular processes, including actin dynamics, cell cycles, gene transcription, adhesion, and vesicular transport. The relevance and significance of this molecule in human diseases and pathological conditions require investigation, with a focus on the development of atherosclerotic cardiovascular diseases as well as the process of aging.
Acknowledgments
This work was supported by research grants from the Study Group of Molecular Cardiology (Japan), from the Japan Heart Foundation (Japan), from a Japan Heart Foundation/Pfizer Grant for Research on Hypertension and Vascular Metabolism (Japan), and from the Tanabe Medical Frontier Conference (TMFC, Japan) to K. Hirano. This work was supported by an International HDL Research Awards Program grant to S. Yamashita. This work was also supported by grants-in-aid to S. Yamashita (Nos. 11557055 and 10671070) and K. Hirano (No. 13671191) from the Ministry of Education, Science, Sports, and Culture of Japan and a research grant to Y. Matsuzawa from JSPS-RFTF97L00801. We thank Drs Takuya Sasaki and Yoshimi Takai (Department of Molecular Biology and Biochemistry, Osaka University, Osaka, Japan) for providing us with Cdc42 constructs. We gratefully thank Drs Hiroshi Kondo (Tokyo Institute of Gerontology, Tokyo, Japan) and Hiroshi Mizusawa (HSRRB) for providing us with the fibroblast cell lines. We thank Dr Hiroyuki Mizuguchi (National Institute of Health Sciences, Tokyo, Japan) for helpful comments on constructing adenovirus vectors. We thank Akira Yaguchi (Carl Zeiss Inc, Tokyo, Japan) for his skillful technical assistance with FRAP.
Footnotes
Presented in part at the 12th International Symposium on Atherosclerosis, Stockholm, Sweden, July 7–10, 2000.
Received December 5, 2001; revision accepted August 19, 2002.
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