PGA2 induces SMAR1 expression
The negative regulation of Cyclin D1 by SMAR1 and PGA2 are well documented, though the mechanisms reported are different (8
). This prompted us to check the interdependence of these pathways and look for a possible regulation of SMAR1 by PGA2. To evaluate this study, we employed MCF-7 cell line derived from mammary epithelia that is characterized by low invasive potential and induced Cyclin D1 level correlated to reduced SMAR1 expression (14
). MCF-7 cells were treated either with vehicle (ethanol) or PGA2 (0–100 μM). Total RNA was isolated 24 h posttreatment and the cDNA obtained was subjected to RT–PCR and Real time RT–PCR analysis. SMAR1 transcript levels increased upto 45 μM PGA2 treatment, after which a steady-state level of the transcript was observed (A). Quantitation by Real time RT–PCR showed that SMAR1 transcript increased 1.8- to 2-fold at 30–100 μM PGA2 (B). The transcript profile was normalized using β-actin. PGA2-induced SMAR1 protein levels were checked using western blot analysis using 30 and 70 μM PGA2. At these concentrations, the protein expression increased by 3.8- and 4.5-fold respectively, while the protein levels were very low in untreated and vehicle-treated cells (C and D). The steady increase in the protein amounts were then visualized using confocal microscopy. MCF-7 cells were treated with PGA2 (15, 30 and 70 μM) and the protein levels were monitored using SMAR1 antibody. A steady increase in the amount of SMAR1 was observed from 15 to 70 μM PGA2 concentrations (E).
Figure 1. PGA2 induces SMAR1 expression. (A) MCF-7 cells were treated with vehicle (alcohol) and various concentrations of PGA2. Total RNA was obtained from aforementioned samples and used for reverse transcription. SMAR1 and β-actin transcript levels were (more ...)
PGA2 stabilizes SMAR1 mRNA
It is well known that most of the reported prostaglandin-mediated effects occur through alteration in mRNA half-life. Results from the previous section show that PGA2 treatment increased SMAR1 transcript by 2-fold but the protein level by 4-fold, indicating a possible involvement of mRNA stability. To address this issue, half-life of endogenous SMAR1 transcript was verified by blocking cellular transcription using Actinomycin D (4 μg/ml) and cells were collected at indicated time points (A). Total RNA was isolated and subjected to RPA as described in ‘Materials and Methods’ section. As shown in A upper panel, SMAR1 transcript level decreased after 4 h in Actinomycin D-treated samples. Interestingly, upon addition of Actinomycin D and PGA2, we observe that the levels of SMAR1 transcript remained steady till 24 h (B, upper panel). This indicates that PGA2 stabilizes SMAR1 mRNA and maintains the steady-state levels of the transcript. The mRNA available at the given time points were normalized to the actin transcript and quantification represented as bar graph (A and B).
Figure 2. PGA2 stabilizes SMAR1 mRNA. (A) Equal number of MCF-7 cells were seeded in 35 mm dishes and 24 h later treated with 4 μg/ml Actinomycin D. Cells were collected after indicated time points and total RNA isolated using Tri reagent. In case of PGA2 (more ...)
SMAR1 has two 5′ UTR variants in MCF-7 cells
The stability and translation of most of the transcripts are associated with the untranslated regions, both at 5′ and 3′ UTRs. Primers were designed to amplify the sequence from the start of exon1 and a part of exon 2 that was expected to yield an intact SMAR1 5′ UTR of 142 bp (18
). The amplimer of 5′ UTR from the cDNA of MCF-7 cells untreated and treated with PGA2 were checked on a 10% polyacrylamide gel. Interestingly, untreated cells showed a single amplimer (C, lane 1) while in case of PGA2 treatment (30 and 70 μM), there was an additional amplimer (C, lanes 2 and 3). The fast migrating amplimer (lower band) was common to both untreated and PGA2 treated cells, while there was an induction of the slower migrating amplimer (upper band) specifically in PGA2-treated cells. Thus, the upper and the lower bands represent two variants of 5′ UTR. Earlier, we have reported the identification of three clones of SMAR1 from mouse thymocyte cDNA library (19
). Out of these, two clones shared the same open reading frame and same translational reading frame of downstream sequence, but differed only by 18 bases in 5′ UTR. The two clones were denoted as ϕ1 (containing full-length 5′ UTR) and ϕ17 (containing variant 5′ UTR lacking 18 bases). When checked using ϕ1 and ϕ17 template controls (D, lanes 2 and 4), we find that the size of upper band obtained upon PGA2 treatment corresponded to the ϕ1 form (the upper band in C, lanes 2 and 3; D, lane 5) and the lower band in untreated and PGA2-treated cells corresponds to ϕ17 form (the lower band in C, lanes 1–3, D, lane3). This was further verified by cloning the corresponding PCR products in PGEMT-Easy
vector and DNA sequencing. RT–PCR analysis of cDNA from MCF-7 cells (untreated or treated with 70 μM PGA2, lanes 1 and 2, respectively) with primers specific for ϕ1-UTR and ϕ17-UTR forms was performed. The results revealed that upon PGA2 treatment there was no apparent change in the ϕ17 form but there was an increase in the ϕ1 form of SMAR1 (Supplementary Figure S1A). The map of SMAR1 gene and the primers used are depicted in Supplementary Figure S1B.
The responsiveness of 5′ UTR to PGA2 is essential for SMAR1 mRNA stability
Next we checked if the responsiveness of SMAR1 UTR to PGA2 contributed to the stability of SMAR1 transcript. The antisense UTR transcript specific to each form was hybridized with the total RNA from MCF-7 cells untreated or treated with PGA2, as described in ‘Materials and Methods’ section. Protected bands revealed that the ϕ17 form was predominant in untreated cells, unlike the ϕ1 form (almost 3–4-times lower than ϕ17, verified by densitometry). An increase in protection of ϕ1 transcript was observed upon PGA2 treatment while ϕ17 form remained almost unchanged (Supplementary Figure S1C). To validate the transcript profiles of ϕ1-UTR and ϕ17-UTR containing SMAR1, cDNA obtained was subjected to Real time RT–PCR analysis using primers specific to the UTRs and melt curve analysis performed. The transcript profile obtained after Actinomycin D treatment showed that the half-life of ϕ1 was 4–6 h while that of ϕ17 varied between 14–16 h (E). Though the transcript level of ϕ1 remained stable after PGA2 and Actinomycin D treatment, ϕ17 transcript levels started to decline from 12 h, as verified by melt curve analysis (F). Quantification of transcripts revealed that ϕ1-SMAR1 level increased 2-fold at 6 h time point of PGA2 treatment and remained steady till 24 h, while there was a negligible change in ϕ17-SMAR1 level (G). These results point out at the early response of ϕ1-UTR to PGA2 treatment that stabilizes the transcript till 24 h. To further assess the response of the two UTRs to PGA2, we performed reporter assays, where the UTRs from both forms were cloned upstream of gfp gene in pEGFP vector. In case of ϕ1-UTR transfection, we observed a 2–3-fold increase in GFP expression upon PGA2 treatment compared to the untreated cells (H, lanes 3–5). ϕ17-UTR transfection however showed only a minor change in the basal expression of GFP, showing that ϕ1 and not ϕ17 is responsive to PGA2 (H, lanes 7–9). Thus, we demonstrate that in MCF-7 cells, ϕ1 form of SMAR1 and not ϕ17 form responds to PGA2.
ϕ1-UTR stem and loop structure is critical for PGA2-induced complex formation
The natural tendency of RNA is to form highly stable secondary and tertiary structures and the alteration in these structures represent a well-known regulatory mechanism for many cellular processes (20
). To determine the differences in the secondary structure that could implicate a functional significance, we analyzed the ϕ1 and ϕ17 UTR sequences as shown in A. Prediction of the secondary structures of the two UTRs was done using M-fold secondary structure prediction software (21
). After energy minimizations, we observed that ϕ17-UTR lacks a small stem and loop structure, pertaining to those missing 18 bases (B).
Figure 3. The ϕ1 form hosts an extra stem and loop (A) The sequence of ϕ1 and ϕ17 reveals that the 5′ UTRs vary by 18 bases. The missing bases are indicated in bold and the transcription start site indicated in italics. (B) Secondary (more ...)
The major factor that controls mRNA turnover is the association of RNA-binding proteins with sequences forming stable secondary structures. We checked for the association of such proteins with SMAR1 UTR, in the presence and absence of PGA2. RNA EMSA were performed to check for putative nucleoprotein complex formation on ϕ1-UTR. Labeled RNA obtained by transcription of the respective 5′ UTR templates (ϕ1 and ϕ17) were used to perform EMSAs. The binding reactions were performed as described in ‘Materials and Methods’ section. The mixture was then run on 6% native gel and subjected to autoradiography. We could detect three specific nucleoprotein complexes on ϕ1-UTR, in the PGA2-treated lane alone compared to the untreated cell lysate (A, lanes 2 and 3). Upon addition of 50-fold molar excess self-cold competitor, we find a complete abolishment of all three shifted complexes, revealing the specificity of the complex formation (A, lane 4). ϕ17-UTR however failed to form nucleoprotein complex, indicating that the missing 18 bases holds the key determinant to bind to certain factors that in turn govern the stability and translation of SMAR1 (B). To validate this, RNA was transcribed from the ϕ1 template encoding the stem and loop structure (named SL1 hereafter). Using this transcript as probe, EMSAs were performed as described earlier. SL1 also showed nucleoprotein complex formation, comparable to the ϕ1-UTR upon PGA2 treatment (D). Further, competition experiments with 10-fold molar excess cold competitors showed the specificity of the complex (E). To identify the importance of the secondary structure imparted by the sequence, binding assays using two mutants were performed. Mutant 2 had a minor modification of the primary sequence but the secondary structure remained largely unperturbed (where nucleotides outside boxB were modified) while in Mutant 1, the stem–loop structure pertaining to boxB was predicted to be partly disrupted (C). When compared to SL1 (F, lane 2) both Mutant 1 and Mutant 2 showed a reduced complex formation, although to varying degrees. Mutant 1, where the core stem structure was disrupted, lacked the specific complex formation compared to Mutant 2 (F, lanes 4 and 6, respectively). Thus, we emphasize that the secondary structure imparted by these 18 bases to SMAR1–UTR is critical for the complex formation. Further, to identify the number and putative molecular weights of the components of specific nucleoprotein complex, UV cross-linking experiments were performed as described in ‘Materials and Methods’ section. The binding mixture employing SL1 from the intact UTR as probe was subjected to UV cross-linking, resolved on 10% SDS–PAGE gel followed by autoradiography. After subtraction of the molecular weight of the RNA probe (~15 kDa), we find the involvement of three specific proteins of ~90, 40 and 15 kDa in forming the ribonucleoprotein complex (G). Though the identity of these factors remains unknown, we believe that these factors that bind to the stem and loop structure of UTR upon PGA2 treatment could be the key in stabilizing the SMAR1 mRNA, resulting in altered protein levels.
Figure 4. SMAR1 UTR binds to complexes in a PGA2-dependent manner. EMSAs for different UTRs were performed as described in Materials and Methods section. (A) PGA2 induces ribonucleoprotein complex formation (indicated as I, II and III) on SMAR1 5′ UTR probe. (more ...)
The responsiveness of 5′ UTR to PGA2 is essential for Cyclin D1 downregulation
Since SMAR1 is known to possess transcriptional regulatory functions, the implication of the responsiveness of 5′ UTR to PGA2 in vivo was verified. The inverse correlation of Cyclin D1 downregulation and SMAR1 induction was first verified upon treatment with 30 and 70 μM PGA2 (A). The results so far suggest that PGA2 treatment stabilizes ϕ1 mRNA, the product of which results in Cyclin D1 repression. To confirm this, we performed luciferase reporter assays where Cyclin D1 regulation by ϕ1 and PGA2 were compared. Cotransfection of 0.5 and 1 μg of ϕ1 SMAR1 along with Cyclin D1 promoter construct (CD1-luc) revealed that ϕ1 downregulated Cyclin D1 by 1.5- and 2.5-fold, comparable to PGA2-treated samples. Moreover, PGA2 treatment after knockdown of SMAR1 using the specific siRNA showed inefficient downregulation of Cyclin D1 (B). This reveals the importance of SMAR1 produced from ϕ1 transcript in PGA2-mediated repression of Cyclin D1. As discussed earlier, the recruitment of SMAR1 and the associated corepressor complex to Cyclin D1 promoter is well documented, and hence we verified if SMAR1 occupied Cyclin D1 promoter in response to PGA2 (C). We observed the interaction of HDAC1 and SMAR1 upon PGA2 treatment (Supplementary Figure S2A and B). Chromatin immunoprecipitation experiments using MCF-7 cells either untreated or treated with 70 μM PGA2 were performed. Amplification of probe II region in SMAR1 and HDAC1 immunoprecipitated from PGA2 treated cells (30 and 70 μM) showed the recruitment of SMAR1 corepressor complex on Cyclin D1 promoter (D, lanes 2 and 3, 9 and 10,). siRNA-treated, SMAR1-pulled chromatin was used to verify the specificity of the recruitment (D, lanes 6 and 7, 12 and 13). These results are consistent with the direct recruitment of HDAC1 and SMAR1 on Cyclin D1 promoter that is responsible for the observed repressive effects. Upon over expression, SMAR1 is shown to deacetylate the histones at H3K9 and H4K8 loci that is accompanied by dephosphorylation of H3-phospho-ser 10 at Cyclin D1 promoter locus. Therefore, we checked if these modifications persist upon PGA2 treatment or if any additional histone modifications are involved in repression of Cyclin D1. We observed a decrease in acetylation at H3K9 (E, lanes 5 and 6), H4K8 (E, lanes 8 and 9) and phosphorylation of H3p-ser 10 (E, lanes 11 and 12) in PGA2-treated cells compared to mock-treated cells. In addition to this, H3K9 monomethylation status that is well documented to have a role in transcriptional activation was investigated. As shown in E, lanes 14 and 15, PGA2 treatment decreased the methylation of probe II region compared to mock treatment. To establish the repression of transcription brought about by SMAR1, the recruitment of RNAP II on Cyclin D1 promoter locus was studied. Decreased amplification of probe II region in RNAP II-pulled samples compared to mock treated suggests decreased transcription from Cyclin D1 promoter locus (E, lanes 2 and 3). Nonspecific probe III was used as negative control (lanes 17 and 18). Mouse and rabbit IgG served as negative controls (D, lanes 15 and 17).
Figure 5. Responsiveness of SMAR1 5′ UTR is essential for exerting its transcriptional modulatory function. (A) RT–PCR analysis of SMAR1, Cyclin D1, and β-actin transcript levels in PGA2-treated (lanes 2 and 3 at 30 and 70 μM PGA2) (more ...)
Cell cycle arrest function of PGA2 mediated by SMAR1 depends on the 5′ UTR
Previous studies by Bhuyan et al.
) showed that PGA2 exerts growth inhibitory activity in many human cell lines. Consistent with this, we found that PGA2 treatment caused G1 phase arrest by DNA content analysis using flow cytometry. We then checked if this growth inhibitory effect of PGA2 is mediated by SMAR1. Hundred-nanomolar SMAR1 siRNA was transfected in MCF-7 cells and 16 h posttransfection, PGA2 was added to the culture media. Cell cycle analysis was done 24 h post-PGA2 treatment, after checking the knockdown of SMAR1 (data not shown). There was a marked shift of the cells from G1 to S and G2 phase, indicating that PGA2 was no longer able to arrest cells in G1 phase in the absence of SMAR1 (A). The results so far suggest the involvement of PGA2 in stabilizing ϕ1 SMAR1 and it is also clear that SMAR1 is required for PGA2-mediated growth arrest. This prompted us to investigate the difference in growth arrest function of SMAR1, in context of the two UTRs it can host. For this, MCF-7 cells were transiently transfected with equal amounts of ϕ1 or ϕ17-SMAR1 and analyzed for cell cycle progression. DNA content analysis revealed a 1.2–2.5-fold higher number of cells in G1/S phase in case of ϕ1-SMAR1 transfection in comparison to ϕ17- SMAR1 (B). This hints that the growth inhibitory effect of PGA2 is mediated at least partially by ϕ1-SMAR1. To check if the PGA2-mediated induction of SMAR1 specifically leads to Cyclin D1 downregulation, western blot analysis to check the status of other cyclins was performed. The results were then verified using siRNA treatment of SMAR1. The specificity of SMAR1 upregulation upon PGA2 treatment was also verified using siRNA treatment (Supplementary Figure S3A). The status of other cyclins (Cyclin A, B and associated cyclin kinases cdks 4 and 6) was unaltered (Supplementary Figure S3B). This result was corroborated using cDNA microarray results (Supplementary Figure S4).
Figure 6. PGA2 requires SMAR1 for its growth-inhibitory function in MCF-7 cells. (A) MCF-7 cells were analyzed by flow cytometry 24 h post-PGA2 treatment in the presence or absence of SMAR1 siRNA. The treatments done are indicated in the graph. The graph represents (more ...)
Breast cancer-derived cell lines from mammary epithelia primarily express ϕ17 form
To find out if the mechanism of downregulation of SMAR1 by altering the mRNA stability is common to breast cancer-derived cell lines, we screened for the presence of the two forms of SMAR1 UTR in ZR75-1, SKBR-3, T47D and MDA-MB-231. Different cell lines were cultured, RNA isolated and limited cycle RT–PCR analysis was performed. RT–PCR analysis showed that ZR-75-1 failed to show the product that corresponds to ϕ1-UTR form but expresses ϕ17-UTR only (C, lane 2 upper and middle panel). This was similar to MCF-7 cells where a very low amplification of ϕ1-UTR transcript was observed (C, lane 6, upper and middle panel). Other cell lines like MDA-MB-231, SKBR3 and T-47D expressed the ϕ1-UTR form predominantly and ϕ17-UTR form to a lower extent (C, lanes 1, 4 and 5, upper and middle panel). HEK-293 cells, where ϕ1 form is expressed predominantly and ϕ17-UTR is undetectable was used as control (C, lane 3, upper and middle panels). The transcript profile we have obtained is in accordance with our previous result, where we have observed that ZR-75-1 and MCF-7 express very low amount of endogenous SMAR1 protein, whereas other cell lines showed a relatively high SMAR1 expression (14
). Since the UTR profile of ZR-75-1 and MCF-7 cells was similar, we checked the response of this cell line to PGA2 treatment and compared to MCF-7 cell line. Upon 30 μM PGA2 treatment, we found a significant induction of the ϕ1-UTR form compared to the untreated cells (D, lanes 1 and 3, upper panel). This was strikingly similar to MCF-7 cells where PGA2 treatment leads to induction of ϕ1-UTR form (D, lanes 2 and 4, upper panel). Cyclin D1 profiles in these cell lines untreated or treated with PGA2 showed that the repression of Cyclin D1 occurred in case of PGA2-treated samples that correlate with the emergence of ϕ1-UTR form.