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Endocrinology, doi:10.1210/en.2005-1426
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Endocrinology Vol. 147, No. 6 3123-3132
Copyright © 2006 by The Endocrine Society

DHCR24-Knockout Embryonic Fibroblasts Are Susceptible to Serum Withdrawal-Induced Apoptosis Because of Dysfunction of Caveolae and Insulin-Akt-Bad Signaling

Xiuli Lu, Fukushi Kambe, Xia Cao, Taemi Yoshida, Sachiko Ohmori, Kohji Murakami, Takahide Kaji, Takehisa Ishii, David Zadworny and Hisao Seo

Department of Endocrinology and Metabolism (X.L., F.K., X.C., T.Y., S.O., H.S.), Division of Molecular and Cellular Adaptation, Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601, Japan; Mitsubishi Pharma Corp. (K.M., T.K., T.I.), Yokohama 227-0033, Japan; and Department of Animal Science (D.Z.), McGill University, Montréal, Québec, Canada H9X 3V9

Address all correspondence and requests for reprints to: Fukushi Kambe, M.D., Ph.D., Department of Endocrinology and Metabolism, Division of Molecular and Cellular Adaptation, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. E-mail: kambe{at}riem.nagoya-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The DHCR24 gene encodes an enzyme catalyzing the last step of cholesterol biosynthesis, the conversion of desmosterol to cholesterol. To elucidate the physiological significance of cholesterol biosynthesis in mammalian cells, we investigated proliferation of mouse embryonic fibroblasts (MEFs) prepared from DHCR24–/– mice. Both DHCR24–/– and wild-type MEFs proliferated in the presence of serum in culture media. However, the inhibition of external cholesterol supply by serum withdrawal induced apoptosis of DHCR24–/– MEFs, which was associated with a marked decrease in the intracellular and plasma membrane cholesterol levels, Akt inactivation, and Bad dephosphorylation. Insulin is an antiapoptotic factor capable of stimulating the Akt-Bad cascade, and its receptor (IR) is enriched in caveolae, cholesterol-rich microdomains of plasma membrane. We thus analyzed the association of IR and caveolae in the cholesterol-depleted MEFs. Subcellular fractionation and immunocytochemical analyses revealed that the IR and caveolin-1 contents were markedly reduced in the caveolae fraction of the MEFs, suggesting the disruption of caveolae, and that large amounts of IR were present apart from caveolin-1 on plasma membrane, indicating the uncoupling of IR with caveolae. Consistent with these findings, insulin-dependent phosphorylations of insulin receptor substrate-1, Akt, and Bad were impaired in the cholesterol-depleted MEFs. However, this impairment was partial because treatment of the MEFs with insulin restored Akt activation and prevented apoptosis. Cholesterol supply also prevented apoptosis. These results demonstrate that the cellular cholesterol biosynthesis is critical for the activation and maintenance of the Akt-Bad cell survival cascade in response to growth factors such as insulin.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CHOLESTEROL IS AN essential biological molecule in animals. It is a component of cell membrane and myelin, a precursor for steroid hormones and bile acids, and is important for the posttranslational modification of hedgehog proteins (1). Recently it has been established that cholesterol is not only a structural component of cell membrane but also the major constituent of the distinct microdomains of plasma membrane, lipid rafts, and caveolae (2, 3).

Caveolae are cholesterol- and sphingolipid-rich, flask-shaped invaginations in cell surface and are enriched in caveolin oligomers, the major protein constituent of the structure, which is a distinct characteristic from lipid rafts lacking caveolin (2, 3). Three caveolins, caveolin-1, -2, and -3, are identified to date. Caveolin-1 and -2 are ubiquitously expressed, whereas caveolin-3 is specifically expressed in muscle cells. The principal role of caveolin-1 in caveolae formation was demonstrated by its knockout mice in which the cells are entirely devoid of caveolae (4). Caveolin-1 can directly bind with cholesterol (5), possibly maintaining the critical concentration of cholesterol required for invagination. Although some functions of caveolae remain controversial, their involvement in endocytosis/transcytosis, cholesterol transport, regulation of signal transduction, and tumorigenesis has been reported (2, 6, 7, 8, 9).

As for the regulation of signal transduction, various signaling molecules and growth factor receptors are shown to be recruited into caveolae by caveolins that, through their scaffolding domain, interact with these molecules (3). Making the signaling molecules in close contact enables efficient signal transduction, which makes caveolae the gateway for signals entering into cells. Studies using cultured adipocytes demonstrated that the insulin receptor (IR) is present in caveolae (10, 11, 12). The importance of cholesterol in the functional interaction of IR with caveolae was shown by the observation that the removal of cholesterol from plasma membrane by methyl-ß-cyclodextrin (CD) disrupts caveolae structure and impairs insulin signaling (10). In addition, it was also reported that caveolin-1 binds with IR through the scaffolding domain (12). However, there exist several controversial reports showing the absence of IR in caveolae (13, 14).

It has been widely accepted that antiapoptotic effects of growth factors are exerted mainly through the activation of Akt/protein kinase B (15, 16, 17). Insulin activates Akt via phosphorylation of insulin receptor substrate (IRS) and subsequent activation of phosphatidylinositol 3'-kinase (PI3K). Tyr-608 residue in rat and mouse IRS-1 (Tyr-612 residue in human IRS-1) is directly phosphorylated by IR. The generation of phosphatidylinositol 3, 4, 5-trisphosphate at the plasma membrane by PI3K recruits Akt, phosphoinositide-dependent kinase (PDK)-1, and PDK2 into proximity, in which Thr-308 and Ser-473 in Akt are phosphorylated by PDK1 and PDK2, respectively. Both phosphorylations are required for full activation. Molecular identification of PDK2 is still not achieved, but there are several candidate kinases (18). Akt is also a serine/threonine protein kinase and phosphorylates various proteins, one of which is Bad, a proapoptotic, Bcl-2 family protein. Unphosphorylated Bad forms a complex with Bcl-XL and induces the release of cytochrome C from mitochondria, triggering apoptosis (19). When Bad is phosphorylated at Ser-136 by Akt, it is sequestrated in cytoplasm by binding with 14-3-3 protein, resulting in cell survival.

We previously cloned the DHCR24 (hDiminuto/Seladin-1) gene, which is abundantly expressed in the cortisol-producing adrenocortical adenoma, compared with the adjacent normal tissue (20). The reduced expression of DHCR24 gene is associated with the increased apoptosis of adrenocortical cells, suggesting that DHCR24 serves as an antiapoptotic protein. Similar antiapoptotic function was suggested in the brain, in which DHCR24 expression is reduced in the regions affected by Alzheimer’s disease (21). Furthermore, DHCR24 was shown to interact with p53 and induce its accumulation (22). On the other hand, it was shown that the DHCR24 gene encodes an enzyme, 3ß-hydroxysteroid-{Delta}24 reductase, which catalyzes the last step of cholesterol biosynthesis, the conversion from desmosterol to cholesterol (23). Therefore, DHCR24 is a multifunctional protein possessing cholesterol-synthesizing and antiapoptotic activities. However, the molecular bases for the antiapoptotic activity are not fully understood.

DHCR24 knockout mice were generated and found unexpectedly to be viable (24) because its genetic abnormalities in human are associated with severe anomalies in development (23). In the present study, to define the physiological significance of de novo cholesterol biosynthesis in cell functions and also elucidate the roles of DHCR24, we investigated the proliferation of mouse embryonic fibroblasts (MEFs) prepared from the knockout mice (DHCR24–/– MEFs). We found that DHCR24–/– MEFs were more sensitive to apoptosis induced by serum withdrawal than wild-type MEFs. It was also found that the cellular cholesterol content was important for the activation and maintenance of the Akt-Bad signaling cascade in response to growth factors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation and culture of MEFs
C57BL/6 x 129SvEv mice with a DHCR24-knockout allele (heterozygous mice, +/–) were provided by Quark Biotech Inc. (Fremont, CA). Several lines of MEFs were prepared from fetuses of gestational age 17–20 d and cultured in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin, streptomycin, and nonessential amino acids (Invitrogen, Carlsbad, CA). The MEFs of 10–20 passages were used for the experiments. In some experiments, MEFs were treated with 1 µg/ml insulin, 2% CD (Sigma-Aldrich, St. Louis, MO) and cholesterol-loaded CD (CLCD). Preparation of CLCD was as follows (25). Cholesterol (200 mg, Sigma-Aldrich) and CD (1 g) were dissolved in 1 ml chloroform and 2 ml methanol, respectively. A 0.45-ml aliquot of the cholesterol solution was added to the CD solution (90 mg cholesterol, 1 g CD), which was stirred and placed for 24 h under a stream of nitrogen gas to evaporate chloroform and methanol. CLCD powder was used at 0.5 mg CLCD per 1 ml serum-free DMEM. The cells were also cultured in the serum-free DMEM containing 40 µg/ml cholesterol, which was prepared by diluting the stock cholesterol solution (20 mg/ml in ethanol). The cell images were obtained with a phase-contrast microscope (IMT-2; Olympus, Tokyo, Japan) equipped with a digital microscope camera (PDMC II; Polaroid, Waltham, MA). Cell proliferation was assessed by water-soluble tetrazolium (WST)-1 method (cell counting kit; Dojindo, Kumamoto, Japan) as described in our previous report (26).

Genotyping and detection of DHCR24 expression
Genotypes and DHCR24 expression were examined by PCR using genomic DNA and RT-PCR using total RNA, respectively. The primer sequences are as follows: Ex1S primer, 5'-TCGCTGGCCGTGTGCGCGCT-3'; Int1AS primer, 5'-ATCCCCACTCCCACGCCCATT-3'; NeoS primer, 5'-GATGGATTGCACGCAGGTTC-3'; mDHCR24S primer, 5'-CACAGGCATCGAGTCATCGT-3'; and mDHCR24AS primer, 5'-GGCACGGCATAGAACAGGTC-3'. The last two primers were used for RT-PCR.

Detection of DNA fragmentation in apoptosis
Both adherent and detached cells in two 10-cm dishes were pooled and washed with PBS. The cells were lysed in 100 µl of cell lysis buffer [10 mM Tris-HCl (pH 7.4), 10 mM EDTA, 0.5% Triton X-100] for 10 min on ice and centrifuged at 12,000 rpm for 20 min to separate intact genomic DNA. The supernatant was treated with 0.4 mg/ml RNase A for 1 h at 37 C, followed by treatment with 0.4 mg/ml proteinase K for 1 h at 37 C. After precipitation with isopropanol and NaCl, the fragmented DNA was resuspended in 20 µl of 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (pH 8.0) and electrophoresed in a 2% agarose gel. DNA fragmentation was also analyzed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) method using an in situ apoptosis kit (Takara, Otsu, Japan) according to the manufacturer’s protocol (20).

Determination of cellular cholesterol and desmosterol contents
Lipid was extracted from MEFs cultured in a 150-mm dish by the Bligh-Dyer method (27). After dried, it was dissolved in ethanol and subjected to HPLC together with the standard cholesterol and desmosterol. All chemicals used were of analytical-reagent grade. Liquid chromatograph (model LC-10A VP and LC-10A, Shimadzu, Kyoto, Japan) was equipped with a variable-wavelength UV-visible detector (model SPD-M10A VP and SPD-10AV; Shimadzu), which was set to monitor the absorbance at 210 nm. The samples were separated through a reversed-phase chromatographic column (TSK gel ODS-80Ts QA, 150 x 4.6 mm inner diameter, particle size 5 µm; Tosoh, Tokyo, Japan). The column temperature was maintained at 40 C. All analyses were carried out isocratically using acetonitrile-methanol as the eluent at a flow rate of 1.0 ml/min. Chromatograms were analyzed with a Shimadzu model CLASS-VP (version 5.04) software.

Western blot analysis
Procedures for preparation of whole-cell lysates and Western blot analysis were described in our previous report (28). In brief, whole-cell lysates (50 µg/lane) were separated by 15% SDS-PAGE and transferred onto polyvinylidene difluoride membrane (Amersham Pharmacia, Piscataway, NJ). The blots were probed with the first antibodies as described below, followed by incubation with horseradish peroxidase-conjugated antirabbit IgG. Rabbit anti-phospho-Akt (Ser473), anti-phospho-Akt (Thr308), and anti-Akt antibodies were purchased from Cell Signaling (Beverly, MA). Rabbit anti-phospho-Bad (Ser136), anti-caveolin-1 and anti-IR ß-subunit antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-phospho-IRS-1 (Tyr608 in rat and mouse IRS-1, Tyr612 in human IRS-1) was from BioSource International (Camarillo, CA). Rabbit antiactin antibody was from Sigma-Aldrich. The proteins were visualized using enhanced chemiluminescence reagents (Pierce, Rockford, IL). The images of the blotted membranes were obtained using an LAS-1000 luminoimage analyzer (Fuji Film, Tokyo, Japan), and densitometric analysis was performed using a software in LAS-1000. The phospho-protein levels were normalized by the actin levels.

Preparation of caveolae
Caveolae fractions were prepared using a detergent-free method (29). Briefly, cell homogenates (postnuclear supernatant fraction) prepared from MEFs in 10 10-cm diameter dishes were layered on 30% Percoll (Amersham Pharmacia) and centrifuged at 84,000 x g for 30 min. The visible band was collected and designated plasma membrane fraction. After sonication, the fraction was reconstituted in 25% OptiPrep (Life Technologies, Inc., Grand Island, NY), placed at the bottom of tube, and overlaid sequentially with 20, 15, and 10% OptiPrep solutions, followed by centrifugation at 56,000 x g for 90 min. The top 5 ml (around half volume of the tube) was collected and reconstituted to 30% OptiPrep solution. This was again placed at the bottom of tube, and 5% OptiPrep solution was layered, followed by centrifugation at 52,000 x g for 90 min. The band in the 5% OptiPrep overlay was collected and designated caveolae fraction. To isolate caveolae-enriched fractions, sucrose gradient centrifugation was also performed using a detergent-free method (30). MEFs in two 10-cm dishes were washed with ice-cold PBS, scraped into 500 mM sodium carbonate buffer (pH 11.0), homogenized, and sonicated. The homogenate was mixed with an equal volume of 90% sucrose solution in 25 mM 2-(N-morpholine) ethane sulfonic acid (pH 6.5) and 150 mM NaCl, placed at the bottom of the tube, and overlaid with 35 and 5% sucrose solutions in the 2-(N-morpholine) ethane sulfonic acid buffer containing 250 mM sodium carbonate. The gradient was centrifuged at 39,000 rpm for 20 h, and 1-ml fractions were collected from the top of the gradient. Proteins were precipitated with trichloroacetic acid and subjected to Western blot analysis.

Determination of total sterol contents
Lipid was extracted from the plasma membrane fractions prepared by ultracentrifugation using 30% Percoll as described above. Total sterol contents in the lipid were measured by an enzymatic cholesterol assay kit (Roche Diagnostics, Mannheim, Germany), which determines the levels of 3ß-hydroxysteroids.

Immunocytochemical analysis
Plasma membrane sheets were prepared as described previously (31). MEFs cultured on glass coverslips were washed in PBS, followed by treatment with 0.5 mg/ml poly-L-lysine in PBS for 20 sec. The cells were then swollen by incubation for 20 sec in a hypotonic buffer [1:3 dilution of buffer A (70 mM KC1, 5 mM MgCl2, 3 mM EGTA, 30 mM HEPES [pH 7.5])], placed in buffer A, and immediately broken open by placing under an ultrasonic microprobe for 2 sec. The membrane sheets were fixed in 2% paraformaldehyde for 20 min and blocked with 1% BSA. They were incubated with a mixture of rabbit anti-caveolin-1 polyclonal antibody (Santa Cruz) and mouse anti-IR ß-subunit monoclonal antibody (BD Biosciences, Bedford, MA) for 60 min and then incubated with a mixture of antimouse IgG antibody conjugated with Alexa fluor-488 (Molecular Probes, Eugene, OR) and antirabbit IgG antibody conjugated with Alexa fluor-568 (Molecular Probes) for 1 h.

Images were obtained using confocal laser microscope (LSM510; Carl Zeiss, Jena, Germany) with a x63 objective, oil immersion lens (numerical aperture = 1.4). The LSM510 was equipped with an argon/krypton laser, which was used at 75% intensity of total 30 mW, with all lines open. For the detection of Alexa fluor-488 fluorescence, the main beam splitter for excitation, the secondary beam splitter for emission, and barrier filter were 488, 570, and 505 nm long pass, respectively. For Alexa fluor-568, they were 568, 570, and 585 nm long pass, respectively. The optical parameters including photomultiplier voltages were the same among the experiments. The spatial resolution was higher than 0.2 µm. The images were obtained by the single laser line, and they were merged by Adobe Photoshop software (version 7.0.1; Adobe Systems Inc., San Jose, CA). Three independent experiments on each of wild-type and DHCR24–/– MEFs were performed, and more than five images were randomly captured from a single experiment with the same set of optical parameters. The membrane integrity was examined by propidium iodide (PI) staining. The cells were treated with 500 ng/ml PI for 5 min. After fixation, PI-positive cells were counted using a fluorescence microscope.

Statistical analysis
Statistical analysis was performed with ANOVA followed by Bonferroni’s multiple t test, and P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Serum withdrawal induces apoptosis of DHCR24–/– MEFs
Genotypes of MEFs prepared from wild-type, DHCR24-knockout (DHCR24–/–) and heterozygous (DHCR24+/–) fetuses were determined by PCR using genomic DNA and three primers, Ex1S primer, Int1AS primer, and NeoS primer, in a single amplification. Because the exon 1 including a translation start site was displaced with a LacZ (ß-galactosidase)/neomycin selection cassette in the DHCR24-knockout locus, the exon 1 DNA fragment of 310 bp is amplified only from the normal allele by PCR using the Ex1S primer (the sense primer in exon 1) and Int1AS primer (the antisense primer in intron 1), whereas the DNA fragment of 969 bp is amplified from the knockout allele by PCR using NeoS primer (the sense primer in the neomycin gene) and Int1AS primer. As shown in Fig. 1AGo, only the 310-bp DNA fragment was amplified from the genomic DNA of wild-type MEFs, whereas both 310- and 969-bp fragments were from the heterozygous DHCR24+/– MEFs. DHCR24–/– MEFs displayed only 969-bp fragment, demonstrating the disruption of the DHCR24 gene in both alleles. Accordingly, the expression of DHCR24 mRNA was detected by RT-PCR in wild-type MEFs but not in DHCR24–/– MEFs as shown in Fig. 1BGo.


Figure 1
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  		FIG. 1. Serum withdrawal induces the cell death of DHCR24–/– MEFs. A, Genotypes of wild-type (W), heterozygous (+/–), and knockout (–/–) MEFs were determined by PCR using genomic DNA. The DNA fragment of 310 bp including a part of the exon 1 in the DHCR24 gene is amplified from the normal allele, whereas the fragment of 969 bp including a part of the LacZ/neomycin selection cassette is amplified from the knockout allele. MW, Molecular weight markers. B, The expression of DHCR24 mRNA in wild-type (W) and knockout (–/–) MEFs was determined by RT-PCR. MW, Molecular weight markers. C, Wild-type (WT) and DHCR24–/– MEFs (104/well) were seeded in 96-well plates on the day before d 0 and cultured in the presence of 10% FBS. The numbers of viable cells were assessed by the WST-1 method on d 0, 2, and 4. Values are expressed as mean ± SD (n = 8). *, P < 0.05 vs. WT on d 4. Similar results were obtained from two separate experiments. D, Wild-type (WT) and DHCR24–/– MEFs were cultured in the absence of FBS. The cell images were obtained with a phase-contrast microscope at 0, 9, and 12 h after the serum withdrawal.

 
Using wild-type and DHCR24–/– MEFs, we first studied their proliferation in the presence of FBS in culture media. As shown in Fig. 1CGo, WST-1 assay revealed that both MEFs could proliferate, but the growth rate of DHCR24–/– MEFs was significantly lowered on d 4 when compared with that of wild-type MEFs, suggesting that de novo cholesterol biosynthesis is required for full proliferation of MEFs. In contrast, when FBS was removed from culture media, a number of DHCR24–/– MEFs were detached within 9 h after the serum withdrawal, whereas the number of the adherent wild-type MEFs appeared to be not altered within 12 h (Fig. 1DGo). Similar results were obtained from other independent lines of DHCR24–/– MEFs (data not shown).

To determine whether the cell death of DHCR24–/– MEFs was due to apoptosis, both adherent and detached cells cultured without FBS for 12 h were collected and subjected to DNA fragmentation assay. As shown in Fig. 2AGo, the fragmented genomic DNA was detected in DHCR24–/– MEFs but not wild-type MEFs. DNA fragmentation in the adherent DHCR24–/– MEFs was detected by TUNEL assay as early as 1 h after the serum withdrawal (Fig. 2BGo). These results demonstrated that DHCR24–/– MEFs were highly susceptible to the apoptosis induced by the serum withdrawal.


Figure 2
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  		FIG. 2. Serum withdrawal induces apoptosis of DHCR24–/– MEFs. Wild-type (WT) and DHCR24–/– MEFs were cultured in the absence of FBS. At 12 h after its removal, adherent and detached cells were collected, and the fragmented genomic DNA was prepared and subjected to agarose gel electrophoresis together with molecular weight markers (MW, A). The DNA fragmentation was also analyzed by the TUNEL method at 0 and 1 h after the serum withdrawal (B).

 
Serum withdrawal impairs Akt-Bad signaling
It has been reported that Akt activity is necessary and sufficient for cell survival (15). We thus studied the phosphorylation statuses of Akt-S473 and Bad-S136 in MEFs after serum withdrawal. As shown in Fig. 3Go, the withdrawal did not markedly alter the phosphorylation status of Akt-S473 and total amount of Akt in wild-type MEFs. The phosphorylation of Bad-S136 was also not altered. Of note, the Akt and Bad phosphorylation lasted for 18 h, despite the absence of growth factors in culture media. In contrast, a rapid and significant dephosphorylation of Akt-S473 within 30 min after the withdrawal was observed in DHCR24–/– MEFs. Bad-S136 was also rapidly dephosphorylated. These results suggested that the reduced Akt activation after serum withdrawal resulted in the apoptosis of DHCR24–/– MEFs.


Figure 3
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  		FIG. 3. Rapid dephosphorylation of Akt and Bad after serum withdrawal in DHCR24–/– MEFs. Wild-type (WT) and DHCR24–/– MEFs were cultured in the absence of FBS for various lengths of time. Whole-cell lysates (50 µg/lane) were subjected to Western blot analysis using anti-phospho-Akt (P-S473), anti-Akt (Total), anti-phospho-Bad (P-S136), and antiactin antibodies. The proteins were visualized using the enhanced chemiluminescence method. The representative results are shown (A). The phospho-Akt and phospho-Bad levels were normalized by the actin levels and are expressed as percentage of the levels of time 0 (B) (n = 4, mean ± SD). *, P < 0.05 vs. the levels of time 0.

 
The FBS used in this study contained approximately 400 µg/ml cholesterol. Because the serum withdrawal induced the apoptosis of DHCR24–/– MEFs but not wild-type MEFs, a decrease in the cellular cholesterol content was suggested to cause the rapid dephosphorylation of Akt-S473. To address this possibility, DHCR24–/– MEFs were cultured for 30 or 60 min in the serum-free media containing cholesterol or CLCD, and the phosphorylation status of Akt-S473 was determined (Fig. 4Go, A and B). It was found that the cholesterol supplementation significantly prevented the serum withdrawal-induced dephosphorylation of Akt-S473. We then measured total sterol contents in plasma membrane fractions (Fig. 4CGo). Serum withdrawal for 60 min significantly decreased the sterol content in the fraction of DHCR24–/– MEFs. CD, which extracts sterols from plasma membrane, also significantly decreased the content. Treatment of the cells with CLCD prevented the loss of sterol sterols from plasma membrane. These results showed the rapid decrease of sterol content in the plasma membrane of serum-starved DHCR24–/– MEFs and its replenishment by CLCD. Together, these observations indicated that the Akt activities in MEFs were largely maintained by the signaling pathway(s), which is sensitive to the sterol contents in plasma membrane.


Figure 4
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  		FIG. 4. Supplementation of cholesterol prevents the serum withdrawal-dependent dephosphorylation of Akt. A and B, DHCR24–/– MEFs were cultured for 30 or 60 min in the serum-free media containing 40 µg/ml cholesterol (+ Cho) or 0.5 mg/ml CLCD (+CLCD), and the phospho-Akt and actin levels were determined by Western blot analysis using whole-cell lysates (50 µg/lane). The representative results are shown (A). The phospho-Akt levels were normalized by the actin levels and are expressed as percentage of the levels of time 0 (B) (n = 4, mean ± SD). *, P < 0.05 vs. the levels of time 0; #, P < 0.05 vs. the levels of the serum withdrawal for 30 min. C, DHCR24–/– MEFs were cultured for 60 min in the serum-free media containing 2% CD or 0.5 mg/ml CLCD. The sterol contents in the plasma membrane fractions were determined using an enzymatic cholesterol assay kit, which determines the levels of 3ß-hydroxysteroids so that it cannot distinguish cholesterol and desmosterol (n = 3, mean ± SD). *, P < 0.05 vs. the levels of serum (+); #, P < 0.05 vs. the levels of serum (–).

 
PI staining of DHCR24–/– MEFs cultured without serum for 60 min revealed that the PI-positive cells were 6.7 ± 1.2% (mean ± SD), suggesting that only small number of cells possessed impaired plasma membrane integrity at this time point.

Cellular cholesterol depletion induces uncoupling of IR with caveolae
Insulin is capable of activating the Akt-Bad cascade (15), and its receptor was reported to be present in the cholesterol-rich, plasma membrane microdomains, caveolae (10, 11, 12). We next studied whether the insulin-Akt-Bad cascade was affected by the serum withdrawal. First, the cellular cholesterol and desmosterol contents were measured in wild-type and DHCR24–/– MEFs 14 h after the removal of FBS (Fig. 5Go). The major sterol in wild-type MEFs appeared to be cholesterol in the presence and absence of FBS. Desmosterol was not detected in both conditions. In contrast, in DHCR24–/– MEFs, the similar amounts of cholesterol and desmosterol were detected in the presence of FBS, although their amounts were roughly half of the cholesterol content in wild-type MEFs. The serum withdrawal for 14 h resulted in a marked decrease in the cholesterol content in DHCR24–/– MEFs but not that of desmosterol.


Figure 5
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  		FIG. 5. Serum withdrawal depletes DHCR24–/– MEFs of cholesterol. Wild-type (WT) and DHCR24–/– MEFs were cultured in the presence and absence of serum for 14 h. Lipids were extracted and subjected to HPLC. The cellular cholesterol and desmosterol contents are indicated by closed and open columns, respectively. UD, Undetectable. n = 3, mean ± SD. *, P < 0.05 vs. the cholesterol level of WT in serum (+); **, P < 0.05 vs. the desmosterol level of WT in serum (+); #, P < 0.05 vs. the cholesterol level of WT in serum (–); ##, P < 0.05 vs. the desmosterol level of WT in serum (–); +, P < 0.05 vs. the cholesterol level of DHCR24–/– in serum (+).

 
We next examined the subcellular localization of IR and caveolin-1 in DHCR24–/– MEFs 12 h after the withdrawal. Caveolin-1 was detected as a single band by Western blot analysis using whole-cell lysates of wild-type MEFs (Fig. 6AGo). IR ß-subunit was detected as two bands, the larger one representing an IR precursor. The plasma membrane and caveolae fractions were prepared from wild-type and DHCR24–/– MEFs cultured without FBS for 12 h by a detergent-free method (29). As shown in Fig. 6BGo, Western blot analysis revealed that the contents of caveolin-1 and IR in the plasma membrane fraction were similar between wild-type and DHCR24–/– MEFs. Both proteins were also detected in the caveolae fraction, indicating the localization of IR in caveolae. In contrast to the plasma membrane fraction, the contents of caveolin-1 and IR in the caveolae fraction appeared to be decreased in DHCR24–/– MEFs when compared with those of wild-type MEFs. The equal loadings of proteins were shown by Coomassie blue staining of the gel (supplemental Fig. 1Go, published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org).


Figure 6
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  		FIG. 6. Cellular cholesterol depletion reduces caveolin-1 and IR contents in caveolae fraction. A, Whole-cell lysates prepared from wild-type MEFs were subjected to Western blot analysis using anti-caveolin-1 and anti-IR antibodies. Molecular weight markers (kilodaltons) are indicated. Cav1, Caveolin-1; IR, IR ß-subunit; IRp, insulin receptor precursor. B, Wild-type (WT) and DHCR24–/– (–/–) MEFs were cultured in the absence of FBS for 12 h. The plasma membrane (5 µg protein/lane) and caveolae (0.5 µg protein/lane) fractions prepared by a detergent-free method (see Materials and Methods) were subjected to Western blot analysis. The result of Coomassie blue staining of the gel is shown in supplemental Fig. 1Go. C and D, Wild-type (WT) and DHCR24–/– MEFs were cultured in the presence or absence of FBS for 12 h. In some experiments, the cells were cultured without FBS for 10 h and then treated with 2% CD for 2 h. Whole-cell lysates were subjected to sucrose gradient centrifugation (see Materials and Methods). Ten fractions (each 1 ml) were collected from the top of the gradient. The proteins were precipitated with trichloroacetic acid and subjected to Western blot analysis. The representative results are shown (C). The results of densitometric analysis on fraction no. 5 are presented (D). The caveolin-1 and IR levels are expressed as percentage of the levels of WT in FBS (+) (n = 3, mean ± SD). *, P < 0.05 vs. the levels of DHCR24–/– MEFs cultured with FBS (+).

 
To confirm this change, subcellular fractionation was performed by a detergent-free method, which uses a sodium carbonate buffer instead of a detergent (30). Wild-type and DHCR24–/– MEFs were cultured with or without FBS for 12 h. In some experiments, the cells were treated with CD extracting sterols from plasma membrane. The cell homogenates were subjected to sucrose gradient centrifugation. Ten fractions were collected from the top layer in the centrifuge tube, and the proteins in these fractions were precipitated and subjected to Western blot analysis. As shown in Fig. 6Go, C and D, caveolin-1 was mainly detected in no. 5 fraction, indicating that this fraction includes caveolae. In wild-type MEFs, the serum withdrawal did not change the caveolin-1 content in the fraction. However, the CD treatment markedly decreased the content, demonstrating the essential role of the membrane cholesterol in maintenance of caveolae structure. In contrast to wild-type MEFs, the caveolin-1 content in no. 5 fraction was significantly decreased by serum withdrawal in DHCR24–/– MEFs, indicating the decreased caveolae number by cellular cholesterol depletion. Similar results were obtained from the IR distribution. In the presence of FBS, IR was mainly detected in no. 5 fraction of both wild-type and DHCR24–/– MEFs, confirming the localization of IR in caveolae. Concomitant with the decrease in the caveolae number by cholesterol depletion, the IR content in the fraction was significantly reduced in DHCR24–/– MEFs.

We next performed immunocytochemical analysis using plasma membrane sheets to study the distribution of caveolin-1 and IR on the membrane. The membrane sheets were prepared from wild-type and DHCR24–/– MEFs cultured without FBS for 12 h. The anti-caveolin-1 and anti-IR antibodies used in this study recognize the intracellular, N-terminal region of caveolin-1, and the intracellular, C-terminal region of the ß-subunit, respectively, which may facilitate the observation from the inside of the membrane. As shown in Fig. 7Go, the fluorescent signals of caveolin-1 from wild-type MEFs were detected as red spots 0.1–0.2 µm in diameter. This is consistent with the previous report that the sizes of typical caveolae and caveolae clusters are 0.1–0.5 µm by the observation with fluorescent microscope (10). The IR signals were detected as green spots. The superimposed image showed that most of the fluorescent signals became yellow, indicating the localization of IR in caveolae. In contrast, the caveolin-1 and IR of DHCR24–/– MEFs were detected as relatively weak and diffuse signals, probably representing the disruption of caveolae structure. In the superimposed image, most of the fluorescent signals looked orange but not yellow, suggesting the reduced IR number in caveolae. In addition, a number of green spots were observed in the superimposed image, indicating the presence of IR apart from caveolae. Taken together, these results demonstrated that the cellular cholesterol depletion impaired the structural integrity of caveolae of MEFs, leading to the uncoupling of IR with caveolae. This could result in the impairment of insulin action.


Figure 7
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  		FIG. 7. Cellular cholesterol depletion induces the uncoupling of IR with caveolae. Immunocytochemical analysis was performed using plasma membrane sheets prepared from wild-type (WT) and DHCR24–/– MEFs cultured without FBS for 12 h. The sheets were probed with rabbit anti-caveolin-1 antibody and mouse anti-IR antibody, followed by a mixture of antimouse IgG antibody conjugated with Alexa fluor-488 and antirabbit IgG antibody conjugated with Alexa fluor-568. Images were obtained using confocal laser microscope. The red and green fluorescent signals represent caveolin-1 and IR proteins, respectively.

 
Immunocytochemical analysis using the plasma membrane sheets prepared from DHCR24–/– MEFs cultured without serum for 30 min was also performed (supplemental Fig. 2A, published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). In the presence of serum, the superimposed image showed that a large number of the fluorescent signals were yellow, indicating the localization of IR in caveolae. In contrast, in the absence of serum for 30 min, the fluorescent signals were weak and diffuse, and there were a few yellow signals, suggesting the reduced number of caveolin-1 and IR, and the disruption of their colocalization. However, in the presence of CLCD, most of signals were yellow, indicating that cholesterol supplementation to the plasma membrane by CLCD prevented the serum withdrawal-induced impairment of caveolae. These results demonstrated that the serum withdrawal induced the rapid and sustained uncoupling of IR with caveolae in DHCR24–/– MEFs.

Caveolae disruption is associated with impairment of insulin action
Wild-type and DHCR24–/– MEFs cultured without FBS for 12 h were treated with insulin for 30 min. As shown in Fig. 8Go, insulin significantly increased the phosphorylation of Akt-S473, Akt-T308, Bad-S136, and IRS-1-Y608 in wild-type cells. In contrast, their phosphorylations were markedly impaired in DHCR24–/– MEFs, demonstrating that the coupling of IR with caveolae was functionally associated with the insulin action in MEFs. Of note, the levels of Akt and Bad phosphorylations in the insulin-treated DHCR24–/– MEFs were comparable with those in wild-type cells without insulin stimulation, which survived for 12 h without FBS, suggesting that the insulin treatment prevents DHCR24–/– MEFs from apoptosis. This possibility was addressed below.


Figure 8
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  		FIG. 8. Cellular cholesterol depletion induces the impairment of insulin action. Wild-type (WT) and DHCR24–/– MEFs were cultured without FBS for 12 h and then treated with 1 µg/ml insulin for 30 min. Whole-cell lysates (50 µg/lane) were subjected to Western blot analysis using anti-phospho-Akt (P-S473), anti-phospho-Akt (T308), anti-Akt (Total), anti-phospho-Bad (P-S136), anti-phospho-IRS-1 (Y608), and antiactin antibodies. The representative results are shown (A). The results of the densitometric analysis are shown (B). The phospho protein levels were normalized by the actin levels, and are expressed as percentage of the levels of WT of insulin (+) (n = 4, mean ± SD). *, P < 0.05 vs. the levels of WT of insulin (–); #, P < 0.05 vs. the levels of DHCR24–/– of insulin (–); +, P < 0.05 vs. the levels of WT of insulin (+).

 
To confirm the importance of the membrane cholesterol in insulin action, we next examined the effects of CD and CLCD on the insulin-dependent Akt activation. As shown in Fig. 9Go, the treatment of wild-type MEFs with CD markedly impaired the insulin-dependent Akt phosphorylation. This impairment was entirely reversed by the subsequent treatment with CLCD. Similar results were obtained from DHCR24–/– MEFs, although the extents of Akt phosphorylation by insulin were less than those in wild-type MEFs.


Figure 9
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  		FIG. 9. Effects of CD and CLCD on the insulin-dependent phosphorylation of Akt. Wild-type (WT) and DHCR24–/– (–/–) MEFs were cultured in the absence of FBS for 3 h [serum(–)], in the presence of 2% CD for 1 h followed by the absence of FBS for 2 h (CD), or in the presence of 2% CD for 1 h followed by the presence of 0.5 mg/ml CLCD for 2 h (CLCD). The cells were then treated with 1 µg/ml insulin for 30 min, and whole-cell lysates (50 µg/lane) were subjected to Western blot analysis using anti-phospho-Akt (P-S473) and anti-Akt (Total) antibodies. The representative results are shown (A). The results of the densitometric analysis are shown (B). The phospho-Akt levels were normalized by the total Akt levels and are expressed percentage of the levels of WT that were cultured without serum and treated with insulin [serum (–) and insulin (+)] (n = 4, mean ± SD). *, P < 0.05 vs. the levels of WT of serum (–) and insulin (–); #, P < 0.05 vs. the levels of WT of serum (–) and insulin (+).

 
The effects of CD and CLCD on total sterol contents in MEFs were studied (supplemental Fig. 2B). The CD treatment significantly decreased the cellular sterol contents in both wild-type and DHCR24–/– MEFs. The subsequent CLCD treatment restored the sterol contents in both MEFs.

Their effects on caveolae structure were also studied by immunocytochemical analysis using plasma membrane sheets prepared from wild-type MEFs (supplemental Fig. 2C). The superimposed images revealed that, even in the absence of serum for 3 h, there were a number of yellow fluorescent signals, suggesting that the de novo cholesterol biosynthesis is sufficient for the maintenance of the membrane cholesterol content and caveolae structure. However, CD treatment markedly decreased the yellow signals, suggesting that CD extracted more cholesterol from plasma membrane than its supply from the de novo biosynthesis. The subsequent CLCD treatment restored the yellow fluorescent signals. These results were consistently associated with the changes in the cellular sterol contents (supplemental Fig. 2B) and the extents of Akt phosphorylation by insulin (Fig. 9Go).

Cholesterol and insulin prevent DHCR24–/– MEFs from apoptosis induced by serum withdrawal
We finally addressed whether cholesterol supplementation or insulin treatment could prevent DHCR24–/– MEFs from the serum withdrawal-induced apoptosis. As shown in Fig. 10AGo, DHCR24–/– MEFs did not undergo apoptosis in the presence of CLCD in culture media, even when they were cultured in the absence of FBS for 9 h. The TUNEL-positive cells were hardly detected (data not shown). Accordingly, Akt-S473 phosphorylation was sustained for 9 h (Fig. 10Go, B and C), indicating the important role of the membrane cholesterol in the maintenance of Akt activity. Insulin alone also prevented the apoptosis by maintaining Akt activation, indicating the partial impairment of insulin action in the cholesterol-depleted MEFs as suggested above (Fig. 8Go). The persistent insulin stimuli were capable of maintaining the Akt activation. When insulin was removed in this condition (supplemental Fig. 3), the phosphorylated Akt levels were decreased as rapidly as those observed in the serum withdrawal (Fig. 3Go), demonstrating the important role of membrane cholesterol in the maintenance of the insulin action.


Figure 10
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  		FIG. 10. Cholesterol and insulin prevent DHCR24–/– MEFs from the serum withdrawal-induced apoptosis. DHCR24–/– MEFs maintained in the presence of FBS [serum(+)] were cultured in the absence of FBS for 9 h [serum(–)], in the presence of 2% CD for 3 h followed by the presence of 0.5 mg/ml CLCD for 6 h (CLCD), or in the presence of 1 µg/ml insulin for 9 h (insulin). The representative cell images obtained using a phase-contrast microscope are shown from three separate experiments (A). The whole-cell lysates (50 µg/lane) were subjected to Western blot analysis using anti-phospho-Akt (P-S473), anti-Akt (Total), and antiactin antibodies (B). The results of densitometric analysis are shown (C). The phospho-Akt levels were normalized by the actin levels and are expressed percentage of the levels of serum (+) (n = 3, mean ± SD). *, P < 0.05 vs. the levels of serum (–).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrated that the lack of the DHCR24 gene increases the susceptibility of MEFs to the apoptosis induced by serum withdrawal (Figs. 1Go and 2Go). The antiapoptotic action of DHCR24 has been suggested in previous reports including ours (20, 21). We showed that the decreased expression of DHCR24 is associated with the apoptosis of adrenocortical cells. Greeve et al. (21) similarly reported its decreased expression in the apoptotic brain areas affected by Alzheimer’s disease. They further reported that the overexpression of DHCR24 protects neuronal cells from apoptosis induced by hydrogen peroxide and ß-amyloid, strongly suggesting that DHCR24 scavenges reactive oxygen species on oxidative stress. Our preliminary data also showed that DHCR24–/– MEFs were susceptible to the hydrogen peroxide-induced cell death, even in the presence of serum in culture media (data not shown). However, this study revealed that the apoptosis of DHCR24–/– MEFs induced by serum withdrawal was due to the absence of cholesterol biosynthesis rather than the lack of the reactive oxygen species-scavenging action.

Several reports demonstrated that IR is localized in caveolae of adipocytes, hepatocytes, and fibroblasts (10, 11, 12, 32). However, some reports showed the existence of IR separate from caveolae (13, 14). The present study demonstrated that IR exists in caveolae of MEFs by using two different, detergent-free methods for caveolae purification and by immunocytochemical analysis using plasma membrane sheets (Figs. 6Go and 7Go). In addition, it was demonstrated that the depletion of membrane cholesterol disrupts the caveolae structure and induces the uncoupling of IR with caveolae (Figs. 6Go and 7Go). This change was associated with the impairment of insulin action, leading to the decreased phosphorylation of IRS-1, Akt, and Bad (Fig. 8Go). It is thus likely that the impaired insulin action is, at least in part, responsible for the apoptosis of cholesterol-depleted DHCR24–/– MEFs.

Not only IR but also PI3K subunits and IRS were reported to localize in caveolae (33, 34). It is thus predicted that caveolae act as a structural platform, which facilitates the efficient coupling of IR with its substrates. Indeed, the present study showed that the coupling of IR with caveolae was associated with the efficient phosphorylation of IRS-1 as well as Akt by insulin (Fig. 8Go).

Recently several candidates for Akt S473 kinase (PDK2) have been reported, which include integrin-linked kinase, partially purified raft-associated S473 kinase, protein kinase CIIß and mammalian target of rapamycin/rictor complex (35, 36, 37, 38). Because the former three kinases are activated at or near plasma membrane, it is possible that the disruption of membrane integrity by cholesterol depletion impairs these kinase activities, resulting in Akt down-regulation. In addition, it was reported that protein phosphatase-1 and -2A directly dephosphorylate Akt (39, 40) and that caveolin-1 binds and inhibits these phosphatases, thereby maintaining the Akt phosphorylation (41). It is thus also possible that the disruption of caveolae results in the activation of the phosphatases and thereby the down-regulation of Akt. However, even if multiple impairments were present in the insulin-Akt-Bad cascade in the cholesterol-depleted cells, they are partial because the persistent stimulation with insulin maintains Akt activation, the levels of which are sufficient for cell survival (Fig. 10Go).

The importance of membrane cholesterol in caveolae structure and function has been demonstrated by using enzymatic cholesterol oxidation and cholesterol-binding reagents such as CD and filipin (42). The present study is the first demonstrating the importance by using the cells devoid of cholesterol biosynthesis. In these cells, the knockout of the DHCR24 gene resulted in the accumulation of desmosterol (Fig. 5Go), which, however, appears to be not as effective as cholesterol in maintaining caveolae integrity. This may due to the decrease in hydrophobicity of desmosterol, which may in turn affect membrane fluidity and thus alter the topology of the basolateral region of caveolae required for the maintenance of caveolae structure (43).

A decrease in cellular cholesterol contents does not seem to be a rare condition as seen in the genetic defects of enzymes involved in the cholesterol biosynthesis. For example, a decease in the membrane cholesterol contents was reported in the hypertrophied adipocytes prepared from obese rats (44, 45). Interestingly, these reports showed that the reduced membrane cholesterol was associated with the altered metabolic activities of the cells including insulin resistance.

In conclusion, the present study demonstrates that the membrane cholesterol levels are critical for the activation and maintenance of the Akt-Bad cell survival cascade in response to growth factors such as insulin. The requirement of cholesterol for cell growth and function has been recognized for many years. However, it is still unclear whether the requirement is just due to the fact that cholesterol is a component for membrane or whether it also plays a regulatory role in cell functions. Several reports addressed this issue and suggested that cholesterol is a specific regulator of cell cycle progression in human neutrophil-like cells (46) and is not displaced with other cholesterol analogs (47). Our observation that the MEFs lacking cholesterol biosynthesis exhibited a marked growth disturbances in the presence and absence of cholesterol in culture media may suggest the regulatory role of cholesterol in cell survival and proliferation.


   Acknowledgments
 
We thank Quark Biotech Inc. and Lexicon Genetics for the generous gift of DHCR24-knockout mice.


   Footnotes
 
This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture, Japan.

The authors have no conflicts of interest.

First Published Online March 2, 2006

Abbreviations: CD, Methyl-ß-cyclodextrin; CLCD, cholesterol-loaded CD; FBS, fetal bovine serum; IR, insulin receptor; IRS, insulin receptor substrate; MEF, mouse embryonic fibroblast; PDK, phosphoinositide-dependent kinase; PI, propidium iodide; PI3K, phosphatidylinositol 3'-kinase; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling; WST, water-soluble tetrazolium.

Received November 9, 2005.

Accepted for publication February 22, 2006.


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Y. Wei, A. T. Whaley-Connell, K. Chen, J. Habibi, G. M.-E. Uptergrove, S. E. Clark, C. S. St