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The Department of Biochemistry and Molecular Biology, and Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina, USA
2Correspondence: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Ave., Charleston, SC 29425, USA. E-mail: ogretmen{at}musc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: longevity assurance gene 1 (LASS1) • ceramide synthase • de novo ceramide generation • sphingolipids • lung cancer
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
.
It is well documented that telomerase is activated in cancer cells and plays an important role in cell immortalization and tumorigenesis. The expression of telomerase results in telomere stabilization and immortality of germ line cells when other oncogenic signals are present (3)
. Recently, expression of a virus-encoded telomerase RNA was shown to promote malignant T cell lymphomagenesis (4)
, demonstrating the role of telomerase in tumor promotion.
Increased telomerase activity is mainly regulated by overexpression of hTERT in the majority of tumors and cancer cells; its overexpression in telomerase-negative cells is sufficient for enzyme activity, suggesting that the activation of hTERT transcription may potentially be the dominant rate-limiting step in telomerase regulation (5)
. A role for telomerase in regulating growth and/or apoptosis of cancer cells independent of the regulation of telomere length has been demonstrated recently (6
7
8)
, suggesting that understanding the mechanisms that control the expression of hTERT will be important for both cancer research and therapy.
The core promoter DNA of hTERT (Fig. 1
) contains recognition sequences for several transcription factors, including Sp1/Sp3 and c-Myc (5)
. Various other transcription factors, such as Ap1 or MZF-2, associate with the hTERT promoter DNA at distant sites and are also involved in regulating its activity (9
, 10)
. However, analysis of DNase hypersensitive regions of chromosomal hTERT DNA in telomerase-positive cells, an indication of a transcriptionally active chromatin structure, identified the core promoter region as the main site for regulating the transcription of hTERT in situ (11)
. These data support the view that identifying mechanisms that control the activity of the core promoter of hTERT is highly relevant to understanding its regulation in human cancer cells.
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Earlier studies have demonstrated a role for bioactive sphingolipid ceramide in the regulation of telomerase in various cancer cell lines (12
, 13)
. Recently, six ceramide synthases (CerS1–6), also known as human homologues of yeast longevity assurance gene 1 (LASS1–6) (14
, 15)
, were identified that specifically regulate the de novo synthesis of endogenous ceramides with a high degree of fatty acid specificity (16)
. For example, CerS1 was shown to specifically generate ceramide with an 18-carbon-containing fatty acid chain (C18-ceramide) whereas CerS6 mainly generates C16-ceramides (14
, 16)
.
It has been well documented that transcription of hTERT is inhibited by ceramide in c-Myc- and JNK-dependent mechanisms (12
, 13)
. Recently, a role for ceramide in repressing the hTERT promoter via deacetylation of Sp3 was revealed in human lung cancer cells (17)
. However, whether ceramides with different fatty acid chain lengths play distinct roles in regulating the hTERT promoter by deacetylation of Sp3, and how acetylated and deacetylated Sp3 regulate the hTERT promoter, are still unknown.
In this study, roles and mechanisms of action of de novo-generated ceramides by CerS in repression of the hTERT promoter via deacetylation of Sp3 by HDAC1 were determined. Novel and mechanistic roles of CerS1-generated ceramide in recruiting deacetylated Sp3/HDAC1 repressor complex to the hTERT promoter, which led to local histone H3 deacetylation and repression of the promoter activity, were also revealed.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; the final concentration of ethanol was 0.01%, which had no effect on cell growth or morphology. Cationic ceramides were dissolved in sterile water.
Promoter and telomerase activity assays
The pGL3-basic plasmid containing hTERT promoter fragment (18)
spanning –279 to +5, which is the minimal functional core promoter designated as pBTdel-279, was kindly provided by Dr. J. C. Barrett (National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA). Transient transfections were performed using the Effectene 228 transfection agent (Qiagen, Valencia, CA, USA) as described previously (17)
. Cells were first transfected with CerS expression vectors for 24 h, then cotransfected with reporter luciferase constructs containing the hTERT core promoter DNA cloned upstream of firefly luciferase, Renilla luciferase cloned downstream of the thymidine kinase promoter (Promega, Madison, WI, USA), or a vetor that contains beta-galactosidase (ß-gal) cDNA for an additional 24 h. The luciferase activity was measured using the Luciferase Assay System (Promega) and a luminometer (Berthold Technologies, Bad Wildbad, Germany) normalized to Renilla luciferase or ß-gal activities, as described by the manufacturer (Promega). The histone deacetylase inhibitors (HDIs), TSA and scriptaid, were used at 100 ng/ml and 2 µg/ml, respectively. The activity of telomerase in cell extracts was measured using a Telomere Repeat Amplification Protocol (TRAP) kit (Invitrogen), as described previously (12)
.
Plasmids and transfections
Full-length wild-type (wt) human Sp3 cDNA cloned in pCMV4 plasmid was kindly provided by Dr. Jon Horowitz (North Carolina State University, Raleigh, NC, USA). The human CerS1, CerS5, the wt, and mutated mouse (m) LAG1/mCerS1, which contains a point mutation at its catalytic site (19)
, cDNAs were obtained from Dr. S. M. Jazwinski (Louisiana State University, Baton Rouse, LA, USA). Human CerS6 was cloned from the total cDNA preparation of UMSCC22A cells, and its mutated form that contains the truncated version of the catalytic domain was generated by a two-round PCR method as described previously (20)
. Transfection of cells was performed using Oligofectamine 228 (Invitrogen) or Effectene 228 (Qiagen, Valencia, CA, USA) as described (17)
. Mutation of the K551of Sp3 was performed by generating point mutations from the wt to arginine (R) or glutamine (Q). The mutations were generated using the Quick change Mutagenesis Kit (Stratagene, San Diego, CA, USA), and mutant sequences were confirmed by direct sequencing at the DNA Sequencing Core Facility (Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, USA).
Ceramide measurements
The endogenous ceramide levels in cellular fractions were measured by high-performance liquid chromatography/mass spectroscopy (LC/MS) as described previously (17)
. The in vitro activity of CerS for the generation of C16- or C18-dihydro-ceramide was performed using microsomal preparation of cells as described previously (21)
.
Immunoprecipitation and immunoblotting
Immunoprecipitation was performed by preclearing 100 µg of cell extract diluted in NETN buffer (20 mM Tris-HCl (pH 8), 0.1% Nonidet P-40, or IGEPAL, 1 mM EDTA and 100 mM NaCl) with protein A/G agarose beads (Santa Cruz Biotech, Santa Cruz, CA, USA) for 1 h at 4°C with end-to-end rotation. The primary antibodies anti-HDAC1 (Santa Cruz), anti-SP3 (Upstate, Lake Placid, NY, USA), or anti-HA-12CA5 (Roche, Nutley, NJ, USA) were added for overnight incubation at 4°C, and the protein–antibody complexes were pulled down using protein A/G agarose beads at 4°C. The beads were washed three times sequentially with 500 µl of 1 x PBS, NETN, 0.1%SDS-NETN, and 10 mM Tris-HCl (pH 8.0) with 0.5% IGEPAL, then resuspended in 10 µl 1 x PBS and 10 µl loading buffer. After incubation at 95°C for 5 min, proteins were separated by SDS-PAGE and examined by Western blot analysis (17)
.
Chromatin immunoprecipitation
Chromatin immunoprecipitation (ChIP) was performed using the ChIP Assay Kit (Upstate), as described previously (17)
.
Histone deacetylase activity
In vitro enzyme activity of HDAC was measured using the Histone Deacetylase Assay kit (Promega, Madison, WI, USA), as described by the manufacturer. In brief, 10 µg of total cell extracts were diluted in HDAC assay buffer and incubated with the fluorescently labeled HDAC substrate peptide at 30°C for 30 min. The HDAC activity was then measured using a Wallac Victor 1420 multilabel counter (Perkin Elmer, Norwalk, CT, USA) with an excitation of 350–380 nm and emission at 440–460 nm, as described by the manufacturer.
Immunofluorescence
Cells grown in Lab-Tek II chamber slides (Nalge-Nunc, Rochester, NY, USA) were fixed with 4% PFA and permeabilized with 0.1% TritonX-100 in 4% PFA. After blocking with 1% BSA, cells were incubated with the primary and secondary antibodies (Jackson Biochemicals, West Grove, PA, USA), then postfixed with 4% PFA. The slides were treated with ProLong Gold Antifade Reagent (Invitrogen) and viewed using the Leica confocal microscope.
Statistical analysis
Experiments were performed in at least three independent trials in duplicate. Error bars were utilized to show SD. Statistical analysis of the data was performed using a Student’s t test, and P < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A, lanes 2–4), their roles in regulating the hTERT promoter and telomerase activity were assessed by reporter luciferase and TRAP assays, respectively. Expression of hCerS1 resulted in increased generation of C18-ceramide by 
2.5-fold (Fig. 2B
) and inhibited the promoter activity of hTERT by 70% (Fig. 2C
) compared with controls. Overexpression of human hCerS5 or hCerS6, which increased the generation of C16-ceramide by 2-fold (Fig. 2D
), had no repressive effect on the activity of the promoter (Fig. 2C
). In addition, while expression of hCerS5 slightly increased, hCerS6 expression resulted in a significant induction of the promoter (by 4.5-fold) compared with controls. Moreover, expression of hCerS1, and not hCerS5 or hCerS6 inhibited telomerase activity by 50% (Fig. 3
A, lane 2) and completely prevented TSA- or scriptaid-induced activation of the promoter (Fig. 3
, panels B, C, respectively).
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The role of CerS1 and C18-ceramide in regulating the hTERT promoter was further evaluated by determining the role of the wt and the mutant mouse mCerS1, which contained a point mutation in its catalytic domain that severely decreased its ability to generate C18-ceramide (Fig. 4
A), in regulating the activity of the hTERT promoter in the absence or presence of TSA. Expression of the mutant mCerS1-FLAG failed to inhibit the promoter activity of hTERT, whereas expression of the wt protein inhibited basal activity by 70% compared with controls and almost completely prevented the activation of the promoter in the presence of TSA (Fig. 4B
). The expression levels of wt and mutant mCerS1 in A549 cells were confirmed by IP, followed by Western blot using the anti-FLAG antibody (Fig. 4B
, lanes 1, 2 in the graph area). Thus, these data show for the first time that LASS1 (CerS1)/C18-ceramide plays a role in inhibiting the hTERT promoter in these cells, whereas CerS5- or CerS6-generated C16-ceramide did not have a repressor effect, suggesting that endogenous ceramides with different fatty acid chain lengths play distinct roles in the regulation of the hTERT promoter.
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Inhibition of the hTERT promoter by LASS1 (CerS1)/C18-ceramide involves the deacetylation of Sp3
It has been shown (17)
that ceramide mediates inhibition of the activity of the hTERT promoter via Sp3, whose deacetylation and repressor functions can be prevented in response to the inhibition of HDAC activity by trichostatin A (TSA), suggesting a role for HDAC in its control. Therefore, to determine whether increased generation of C18-ceramide by hCerS1 plays a role in regulating HDAC, its effects on the in vitro activity of HDAC were examined in total cell lysates using a fluorescently labeled small peptide as a substrate, as described in Materials and Methods. As shown in Fig. 5
A, forced expression of hCerS1 resulted in a slight (30%) but significant increase in the activity of HDAC in vitro when compared with vector controls.
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Next, to investigate whether mechanisms by which hCerS1-generated C18-ceramide regulates the activity of the hTERT promoter involve the deacetylation of Sp3, CerS1 was expressed in A549 cells and its roles in the deacetylation of Sp3 were examined. The data showed that expression of hCerS1 mediated the deacetylation of Sp3 significantly as compared with controls (Fig. 5B
, top panel, lanes 2 and 1, respectively). Total Sp3 protein levels were used as a loading control in these samples (Fig. 5B
, bottom panel). Expression of CerS6 had no effect on the acetylation status of Sp3 (data not shown).
To determine the mechanism involved in ceramide-mediated deacetylation of Sp3, association of Sp3 with HDAC1, which is known to repress the hTERT upon its recruitment to the promoter (22)
, was examined by coimmunoprecipitation, followed by Western blot. The data demonstrated that increased generation of C18-ceramide by hCerS1 markedly increased the interaction between Sp3 and HDAC1 compared with vector-transfected controls (Fig. 5C
, top panel, lanes 2 and 1, respectively). Overexpression of CerS6, however, had no effect in Sp3/HDAC1 association (data not shown), and there was no change in association between HDAC2 and Sp3 in the absence or presence of ceramide (Fig. 5D
).
Increased interaction between Sp3 and HDAC1 in response to LASS1/CerS1 expression was also confirmed using confocal microscopy. As shown in Fig. 5E
, the data revealed that hCerS1 expression (right panel) increased the colocalization of Sp3 (green, top left quadrant) with HDAC1 (red, top right quadrant) in the nucleus (merged yellow, bottom left quadrant) when compared with vector controls (left panel). Taken together, these data suggest that ceramide-mediated repression of the hTERT promoter involves the deacetylation of Sp3, which is concomitant with increased HDAC activity, and induces interaction between Sp3 and HDAC1.
Roles of constitutively acetylated or deacetylated Sp3 mutant proteins in regulating the hTERT promoter in S2 cells
Our previous data (17)
and results presented here demonstrate a role for exogenous C6-ceramide and LASS1/CerS1-generated endogenous C18-ceramide, respectively, in regulation of the hTERT promoter activity via controlling the acetylation/deacetylation status of Sp3. However, precise roles and mechanisms of action of deacetylated vs. acetylated Sp3 in regulating the hTERT promoter are still unknown. Therefore, to assess the specific roles of acetylated and deacetylated Sp3 proteins in regulating the hTERT promoter, several mutations of K551 residue of Sp3, reported to be the single acetylation site of the protein (23)
, were generated. Since the K551R and K551Q mutants are generally designed to mimic constitutively deacetylated and acetylated lysine of proteins, respectively (24)
, point mutations to convert K551 to arginine (R) or glutamine (Q), designated K551R or K551Q, respectively, were introduced separately in Sp3 cDNA with a hemagglutinin (HA) tag on their N termini, as described in Materials and Methods. First, expression of the exogenous wt and mutated Sp3-HA proteins in S2 cells, which do not express endogenous Sp3 or Sp1 (25)
, were confirmed by immunoprecipitation, followed by Western blot using anti-HA and anti-Sp3 antibodies, respectively (Fig. 6
A, lanes 1–3, respectively; lane 4 shows untransfected S2 cell extracts used as negative controls). Cells that expressed HA-Sp3 proteins were transiently cotransfected with reporter plasmids containing the hTERT core promoter DNA cloned upstream of firefly luciferase and Renilla luciferase cloned downstream of the thymidine kinase promoter, which was used for normalization. After that, roles of wt and mutant Sp3 proteins in regulating the hTERT promoter activity were analyzed. As expected, while expression of the K551R mutant was repressed by 50%, the K551Q mutant induced activity of the hTERT promoter by 4.5-fold compared with vector-transfected controls (Fig. 6B
). These data demonstrated that acetylated Sp3 at K551 activates, whereas deacetylated Sp3 at K551 represses, the hTERT promoter.
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In addition, treatment of untransfected S2 cells with TSA, which induces the basal activity of the hTERT promoter, did not cause any significant increase; however, when wt-Sp3 was expressed, there was an 20-fold activation of the hTERT promoter in response to TSA (Fig. 6C
). On the other hand, expression of K551R mutant of Sp3 did not respond to TSA for activation of the promoter in these cells (Fig. 6C
), implying that TSA-induced activation of the hTERT promoter requires acetylated Sp3.
Alterations of the acetylation/deacetylation status of Sp3 play a role in the regulation of the hTERT promoter in A549 cells
Roles of deacetylated and acetylated Sp3 in regulating the hTERT promoter were also determined in A549 cells. First, the acetylation status of the wt and mutant Sp3 proteins was detected using pan-acetyl-lysine antibody, and the data showed that while exogenously expressed wt-Sp3 was slightly acetylated (its acetylation state is much lower than endogenous Sp3 in A549) (17)
, its K551Q and K551R mutants showed much weaker acetylation signals (Fig. 7
A, bottom panel, lanes 1–3). These data support the idea that K551 is the major acetylation site for Sp3, but there appear to be other minor acetylation sites in this protein that are still detectable in K551Q and K551R mutants at low levels (Fig. 7A
, bottom panel, lanes 2, 3). Figure 7A
shows the equal expression of exogenous Sp3 and its mutants in A549 cells (top panel, lanes 1–3) as determined by IP, followed by Western blot.
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To determine whether deacetylation of Sp3 alters its binding to the hTERT promoter DNA in situ, ChIP assays were performed using A549 cells that transiently expressed wt-, K551Q-, and K551R-Sp3 proteins (Fig. 7B
). After cross-linking with formaldehyde, DNA–protein complexes were immunoprecipitated using antibodies that recognize the HA tag (for detecting HA-Sp3 proteins), HDAC1, Sp1, or RNA polymerase II; interaction of these proteins with the chromosomal hTERT promoter DNA were examined as described previously (17)
. As seen in Fig. 7B
, the data showed that K551R-Sp3 strongly associated with the hTERT promoter, whereas K551Q-Sp3, which mimics constitutively acetylated Sp3, completely lost its interaction with the promoter DNA (top panel, lanes 5 and 3, respectively). Remarkably, K551R-Sp3 induced the association of HDAC1 with the hTERT promoter DNA significantly, which was not detectable in cells that expressed K551Q-Sp3 or wt-Sp3 (Fig. 7B
, lanes 3, 2, and 1, respectively). Moreover, deacetylated Sp3 (K551R) markedly prevented interaction of RNA polymerase II with the promoter, whereas the binding of Sp1 and RNA polymerase II was induced in cells that expressed K551Q-Sp3 when compared with controls (Fig. 7B
, lanes 3, 2, and 1, respectively).
The functional roles of K551Q and K551R mutants of Sp3 in regulating the hTERT promoter were also examined using luciferase reporter assays in A549 cells. As shown in Fig. 7C
, expression of the K551R-Sp3 mutant, which mimics constitutively deacetylated protein, significantly reduced the activity of the promoter whereas expression of the K551Q mutant of Sp3 caused a slight increase in the hTERT promoter activity in A549 cells (Fig. 7C
). Taken together, these data demonstrate that while acetylated Sp3 induces, deacetylated Sp3 mediates, the repression of the hTERT promoter in both S2 and A549 human lung cancer cells. These data together with the ChIP analysis shown in Fig. 7B
suggest that deacetylated Sp3, which strongly interacts with the hTERT promoter, induces recruitment of HDAC1 and inhibits the interaction of RNA polymerase II to the promoter, providing a mechanism for the repressor function of deacetylated Sp3.
In addition, to examine the functional roles of deacetylated and acetylated Sp3 in ceramide-mediated regulation of the hTERT promoter, cells that express wt-, K551R-, or K551Q-Sp3 proteins were treated with exogenous C6-ceramide, which has been shown to mediate the deacetylation of Sp3 (17)
, and its effects on the hTERT promoter activity were assessed by luciferase reporter assays. Consistent with previous data (17)
, treatment with C6-ceramide resulted in the inhibition of promoter activity by 70% (Fig. 7C
), which was completely prevented in response to the expression of the constitutively acetylated K551Q-Sp3, suggesting that regulation of acetylation/deacetylation status of Sp3 plays a critical role in ceramide-mediated repression of the hTERT promoter.
Consistent with its repressor roles on the hTERT promoter via deacetylation of Sp3 (17)
, treatment of cells with C6-ceramide also resulted in a rapid association between Sp3 and HDAC1 within 6 h of treatment compared with untreated controls (Fig. 8
A, lanes 3 and 1, respectively). Treatment with C6-ceramide at various times (0–48 h) induced the recruitment of Sp3 and HDAC1 to the hTERT promoter DNA in a time-dependent manner (Fig. 8B
, lanes 1–4). Ceramide also caused a marked reduction in the association of acetylated histone H3 with the promoter DNA within 6 h (70%) compared with controls (Fig. 8B
, lanes 2 and 1, respectively); after 24 and 48 h treatment, acetylated histone H3 that bound to the hTERT promoter DNA was under detectable levels (Fig. 8B
, lanes 3 and 4, respectively), suggesting a role for HDAC1 in histone modification/deacetylation leading to the repression of the hTERT promoter. Remarkably, inhibition of the HDAC1 activity by pretreatment with TSA prevented the recruitment of Sp3 and HDAC1 to the hTERT promoter DNA, which also blocked histone H3 deacetylation in response to ceramide treatment (Fig. 8C
). However, inhibition of the HDAC1 activity by TSA did not have any significant effect on the association of Sp3 with HDAC1 in response to ceramide (data not shown). Thus, these data demonstrate that deacetylation of Sp3 by HDAC1 upon their association is a key step in facilitating their recruitment to the promoter DNA for repression. In fact, increased association of Sp3/HDAC1 and their recruitment to the hTERT promoter in response to C6-ceramide within 6 h, and not 1 h, was also consistent with the time-dependent repression of promoter activity in response to ceramide (Fig. 8D
).
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Taken together, these data suggest that ceramide regulates the repression of the hTERT promoter by mediating the deacetylation of Sp3 via increased association between Sp3/HDAC1, which results in their recruitment to the promoter DNA, leading to local histone modification (histone H3 deacetylation) and repression of the promoter.
This hypothesis was further tested in response to increased generation of de novo-generated C18-ceramide by hCerS1 in A549 cells. Expression of wt-hCerS1 resulted in a significant increase (4-fold) in the recruitment of HDAC1 to the hTERT promoter compared with vector controls, leading to the deacetylation of histone H3 on the promoter (Fig. 9
A, lanes 2 and 1, respectively). On the other hand, expression of its catalytically inactive truncated mutant form did not have any significant effects compared with controls (Fig. 9A
, lanes 3 and 1, respectively), suggesting an important role for the generation of C18-ceramide in this process. The expression levels and catalytic activities of wt and mutant hCerS1 compared with vector controls for generating C16- and C18-ceramides (detected by the incorporation of palmitoyl- or stearoyl-CoA into dihydrosphingosine, respectively) were also confirmed by Western blot and CerS enzyme activity assay (Fig. 9B
, top and bottom panels, respectively).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, treatment with C6-ceramide or generation of C18-ceramide by CerS1 expression led to a rapid association between Sp3 and HDAC1, resulting in deacetylation of Sp3, which then enhanced the recruitment of HDAC1 to the hTERT promoter, leading to local histone H3 deacetylation and repression of the promoter. Thus, these results provide novel mechanistic information about the roles and mechanisms of action of deacetylated Sp3 and HDAC1 in ceramide-mediated regulation of the hTERT promoter.
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Previous studies revealed that yeast longevity assurance gene 1 (LAG1) and its homologue LAC1 function as key components of ceramide synthase (CerS) of the de novo pathway (14)
. LAG1 regulates longevity in yeast such that its deletion prolongs life span (26)
. Previously, the mCerS1 was identified to specifically regulate the generation of C18-ceramide (14
, 16)
. It was shown recently that HNSCC tumors contain lower amounts of C18-ceramide and that defects in CerS-dependent ceramide generation might play important roles in human head and neck squamous cell carcinoma (HNSCC) pathogenesis (27)
. Indeed, overexpression of CerS1 with increased C18-ceramide generation resulted in apoptosis in HNSCC cells in situ. The results presented here also agree with these data, which showed that CerS1 via C18-ceramide generation plays an important role in inhibiting the hTERT promoter in A549 cells, which is a known antiproliferative response (21
, 27)
. However, expression of CerS5 or CerS6, which mainly generate C16-ceramide, did not inhibit the activity of the promoter. These data are intriguing, because in our previous study the data showed that generation of C16-ceramide in fractions enriched in nuclei in response to gemcitabine resulted in inhibition of the hTERT promoter and in deacetylation of Sp3 (17)
. Thus, these results suggest that subcellular localization of endogenous ceramides and their downstream targets might be important in determining their distinct functions in cells. Indeed, specific roles of ceramide in structured membrane microdomains have been described recently (28
, 29)
; whether C18-ceramide localizes to these membrane domains, however, needs to be examined.
Expression of CerS6, which resulted in the generation of C16-ceramide, appeared to induce hTERT promoter activity significantly (see Fig. 2C
and Fig. 3B
), which is consistent with the higher levels of C16-ceramide in the tumor tissues of patients with head and neck (27)
as well as lung cancers (unpublished data) compared with their adjacent normal tissues. The specific roles as well as mechanisms of action of CerS6 and C16-ceramide in the up-regulation of the hTERT promoter activity, however, are still unknown and need to be determined. Recently, a role for ceramide 1-phosphate (C1P) in the enhancement of growth in various models (30
, 31)
, including A549 cells (31)
, has been revealed, and it is possible that C16-ceramide might be a potential reservoir for the generation of prosurvival metabolite C1P by ceramide kinase (32)
in these cells. However, whether C1P plays a role in the activation of telomerase remains unknown.
In addition, ceramide, both endogenous C18-ceramide via CerS1 expression and exogenous C6-ceramide, which is known to be deacylated, then its sphingosine is reacylated to generate endogenous ceramides, including C18-ceramide, by the sphingosine recycling pathway (33)
, increased the association of HDAC1 with Sp3, concomitant with the deacetylation of Sp3. However, whether short chain ceramide exerts its effects on HDAC1/Sp3 association by its recycling to generate C18-ceramide, or whether its subcellular localization and/or downstream targets are similar to that of C18-ceramide, are still unknown and need to be determined. Nevertheless, treatment with exogenous ceramide also helps when examining the kinetics of Sp3/HDAC1 association and its recruitment to the promoter in a time-dependent manner, leading to histone H3 deacetylation and repression of the hTERT promoter.
The data also revealed a role for ceramide in inducing the in vitro activity of HDAC. It is well documented that histone acetyl transferases acetylate nonhistone proteins, and HDACs are also known to deacetylate nonhistone substrates (34)
. In fact, various transcription factors have been identified as substrates of class I HDACs. For example, through interaction with HDAC1, retinoblastoma protein (Rb) recruits the Sin3 (Swi-independent 3) deacetylase complex to E2F (adenovirus E2 promoter binding factor) and UBF (ribosomal promoter upstream binding factor) for their deacetylation (35
, 36)
.
Therefore, these data show a functional role for Sp3/HDAC1 in ceramide-mediated control of the promoter. Inhibition of HDAC activity by TSA prevented recruitment of Sp3 and HDAC1 to the hTERT promoter without altering their interaction with each other (data not shown) in response to ceramide. These data, then, suggest that deacetylation of Sp3 by HDAC activity is a crucial step for their recruitment to the hTERT promoter. However, how ceramide mediates the rapid association between Sp3 and HDAC1 that leads to its deacetylation and how deacetylation of Sp3 increases its interaction with the hTERT promoter DNA in situ are still unclear and need to be determined. It should also be noted that the sequential immunoprecipitation of Sp3, followed by HDAC1, altered the detection of HDAC1/hTERT promoter DNA interaction in our previous study (17)
. However, immunoprecipitation of HDAC1 and Sp3 in separate experiments shown here uncovered the recruitment of HDAC1 to the hTERT promoter DNA in response to hCerS1 and C18-ceramide or C6-ceramide in this study.
These data agree with a previous report demonstrating that recruitment of HDAC1 with Rb as a complex plays a role in repressing the hTERT promoter in response to anchorage deprivation of stratified squamous epithelial cells and squamous cell carcinoma lines (22)
. Sp1/Sp3 was also reported to recruit HDAC to the hTERT promoter, leading to its repression in human somatic cells (37)
, and recent data have shown the repression of hTR expression via c-Jun-NH2-kinase by enhancing its repression through Sp3 (38)
, supporting the importance of Sp3 in the regulation of telomerase.
Moreover, the role for HDIs, such as TSA, in the robust activation of the hTERT promoter has been demonstrated (39)
. Specifically, overexpression of HDAC1 repressed the expression of hTERT and TSA treatment resulted in the hyperacetylation of histones at the hTERT core promoter (39)
. However, a role for Sp3 in this process has been unknown. The data presented here revealed that expression of the K551R-Sp3 mutant, which cannot be acetylated, prevented TSA-induced activation, suggesting a role for acetylated Sp3 in activating the hTERT promoter by HDI. In control Drosophila S2 cells, which do not express any endogenous Sp1 or Sp3, TSA was unable to activate the hTERT promoter, but cells that expressed exogenous wt-Sp3, which was acetylated in these cells (data not shown), TSA significantly induced hTERT promoter activity, suggesting that Sp3 expression and acetylation are necessary for HDI-induced activation. Similar to the hTERT promoter, inhibition of HDAC activity by HDI also activated the expression of TGF-ß receptors type I (40)
.
It should also be noted that there were no other detectable post-translational modifications of Sp3, such as phosphorylation, N-acetylglucosamylation, or sumoylation, in these cells (17)
, although Sp3 was shown to be sumoylated in other cell models (41
, 42)
. Also, ceramide inhibited the activity of the hTERT promoter by 70% in vector-transfected control cells, and its inhibitory function was reduced by 43% when cells that expressed constitutively deacetylated Sp3 mutant (K551R-Sp3) were treated as compared with controls (see Fig. 7C
). These data suggest that although deacetylation of Sp3 is necessary, other mechanisms, such as regulation of Myc (12)
and/or JNK (13)
, are also involved in ceramide-mediated regulation of the hTERT promoter. The involvement of Sp1/Sp3-mediated regulation of the hTERT promoter has been elucidated in an independent study (43)
, recently.
In summary, these data reveal for the first time a mechanism for the Sp3-dependent regulation of hTERT promoter via ceramide signaling involving the interaction of Sp3 with HDAC1, leading to deacetylation of Sp3, which then plays a critical role for the recruitment of HDAC1 to the hTERT promoter, resulting in histone H3 modification and repression (Fig. 10)
. These data also implicate that targeting the ceramide/Sp3/HDAC1 axis might help develop novel strategies for the repression of hTERT, and thus inhibition of telomerase expression.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication March 26, 2007. Accepted for publication May 3, 2007.
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