Ras inhibits TGF‐β‐induced KLF5 acetylation and transcriptional complex assembly via regulating SMAD2/3 phosphorylation in epithelial cells
Abstract
Acetylated Kruppel‐like factor 5 (KLF5) is essential for transforming growth factor‐β (TGF‐β) to properly regulate gene transcription in the inhibition of cell proliferation and tumor growth. Ras oncogenic signaling can convert TGF‐β from a tumor suppressor to a tumor promoter; however, its ability to utilize the KLF5 transcription factor to modulate TGF‐β functions is still unknown. Therefore, in this study, we sought to determine whether Ras signaling altered TGF‐β‐induced KLF5 acetylation and the assembly of the p300‐KLF5‐SMADs transcriptional complex in gene regulation. Not only did we determine that Ras signaling inhibited TGF‐β‐induced KLF5 acetylation and interfered with TGF‐βfunction in p15 induction and Myc repression, but also TGF‐β‐induced SMAD3 C‐terminal region phosphorylation was necessary for TGF‐β to induce KLF5 acetylation. Moreover, Ras activation further interrupted the interactions amongst p300, KLF5, and SMAD4, as well as the binding of p300‐KLF5‐ SMADs complex onto the TGF‐β‐responsive promoter elements for both p15 and Myc. These findings suggested that KLF5 mediated the crosstalk between TGF‐β and Ras signaling, and that suppression of TGF‐β‐induced KLF5 acetylation by Ras activation; this altered TGF‐β‐induced assembly of p300‐ KLF5‐SMADs complex onto gene promoters to convert the function of TGF‐β in gene regulation.
1| INTRODUCTION
Transforming growth factor‐β (TGF‐β), a potent inhibitor of epithelial cell proliferation, acts as a tumor suppressor in the early stage of cancer development.1,2 In late‐stage tumor progression, however, TGF‐β often loses its tumor‐ suppressive activity and acquires a tumor‐promoting activity.3 While this functional conversion is commonin carcinogenesis, the underlying molecular mechanism is still poorly understood.Ras oncogenic signaling can render cell resistance to the inhibitory function of TGF‐β on cell proliferation,4 and the interaction of Ras with TGF‐β can convert it from a tumor suppressor to a tumor promoter, as Rasand TGF‐β collaborate to make cells more motile and invasive.5 Blocking Ras signaling can rescue the inhibitory effect of TGF‐β on cell proliferation.6 During the change of TGF‐β function in tumorigenesis,cytostatic genes such as p15 and Myc lose their normal response to TGF‐β1; however, it is unknown whetherRas reverses the transcription of TGF‐β‐regulated cytostatic genes.SMAD2 and SMAD3 are phosphorylated at their C‐terminal region (CTR) with TGF‐β signal stimulation and form complexes with SMAD4 to translocate in thenucleus and regulate the transcription of target genes.7 The linker region of SMAD2 and SMAD3, on the otherhand, contains both confirmed and potential phosphor- ylation sites for Ras‐related kinases including the ERK MAP kinase, c‐Jun NH2‐termianl kinase, and other kinases.8 The Ras signal suppresses the phosphorylationof SMAD3 at its CTR but stimulates phosphorylation at the linker region.9KLF5 binds to GC boxes at gene promoters to regulate their transcription.10,11 KLF5 has dual functions in the control of gene expression and cell proliferation.11 On one hand, it can promote the proliferation of various types of cells including epithelial and cancer cells,10-12 and its expression is upregulated upon Ras activation.13,14 Diversely, it can also inhibit the proliferation of cancercells15-18 likely as an essential coactivator of the TGF‐βpathway19,20 or through a nonclassical pathway involving interaction with estrogen receptor beta.
KLF5 acetyla- tion at lysine 369 (K369) plays an important role in the function of KLF5 in cell proliferation, tumor growth, andself‐renewal and differentiation of mouse embryonicstem cells.21,22 In prostate cancer cells, KLF5 inhibits their growth with TGF‐β treatment, which leads to the induction of KLF5 acetylation (K369)19,20,23; however,when KLF5 acetylation (K369) is blocked by mutation, it promotes tumor growth.24 Mechanistically, K369 acetyla- tion is essential for KLF5 to regulate the transcription of its target genes. For example, acetylation of KLF5 is indispensable for KLF5 to combine on p21 promoter and form a repressive complex.25 Moreover, acetylation is critical for KLF5 to bind the p15 promoter and regulateits transcription.20 KLF5 binds to MYC‐Miz‐1 complexand functions as a repressor of p15 transcription; however, KLF5 binds to SMAD2/3/4 upon TGF‐β treatment, and then p300 is recruited to their complexand KLF5 is acetylated by p300; thus, KLF5 is reversed to an activator of p15 expression. In addition, acetylated KLF5 is essential for the formation of the p300‐KLF5‐SMADs complex on the promoter of TGF‐β target genes,such as p15 and Myc, and prevention of KLF5 acetylation could reverse the function of TGF‐β and KLF5.19,20,23Therefore, we hypothesize that the interruption of TGF‐βfunction in cell proliferation by Ras activation is mediated by the deacetylation of KLF5, which involves the phosphoryla- tion of SMAD3 at different regions and leads to the deregulation of cytostatic genes. In this research, we tested this hypothesis by examining the expression of p15 and Myc,acetylation of KLF5, and TGF‐β‐induced phosphorylation ofSMAD2 and SMAD3 in Ras‐transformed and control epithelial cells. Furthermore, we analyzed the formation ofTGF‐β‐induced KLF5‐p300‐Smads transcriptional complex and its binding to the promoters of p15 and Myc in the context of TGF‐β and Ras interaction. We found that Ras activation changed the effect of TGF‐β on gene expression through the regulation of KLF5 acetylation and the assemblyof KLF5 transcriptional complex onto gene promoters in epithelial cells.
2| MATERIAL AND METHODS
The HaCaT epidermal epithelial cell line was a gift from Dr. Robert A. Swerlick (Emory University) and it was main- tained following established procedures.26 The COS‐1 andHEK293 cell lines were purchased from the American TypeCulture Collection (ATCC, Manassas, VA). The TGF‐β was from R&D Systems (TGF‐β1; Minneapolis, MN). The sequence of small interfering RNA (siRNA) for KLF5 was 5′‐AAGCTCACCTGAGGACTCA‐3′. The antibody against the KLF5 was previously described.27 Antibodies againstphosphorylated SMAD3 at S204, T179, and S214 were purchased from Abcam Inc (Cambridge, MA). Antibodies against Myc (C‐MYC) and p15 were from Cell SignalingTechnology (Danvers, MA).Retroviral vectors pBabe‐zeo‐Ha‐RasG12V and pBabe‐ zeocin were amiable gifts from Dr. Hiroshi Nakagawa, University of Pennsylvania.28 The retroviruses wereproduced following a previous study.28 For infection, 1× 105 of HaCaT cells were seeded into each well of a six‐ well tissue culture plate and incubated overnight. Themedium was replaced with 2 mL of fresh medium and0.4 mL of virus‐containing medium was then added in the presence of 4 μg/mL of polybrene (Sigma, St Louis, MI). The plate was centrifuged at 1800 rpm at roomtemperature for 30 minutes, and then incubated at 37°C. After trypsinizing and replating, the cells were placed in a1:10 dilution on the third day; selection was initiated with 400 μg/mL of zeocin (Invitrogen) and continued for several weeks until zeocin‐resistant Ras‐transformed cells (HaCaT‐Ras) were established.The procedures were described previously.27
RNA expression was analyzed by real‐time polymerase chain reaction (PCR) while protein expression was detected by Western blotanalysis. The procedure for siRNA transfection was described previously.29The procedure for coimmunoprecipitation was described previously.19 Briefly, cells with different treatments were lysed in lysis buffer, and cell extracts were incubated with antibodies against human p300 (Millipore, Billerica, MA) or SMAD4 (Santa Cruz Biotechnology, Santa Cruz, CA). Precipitated proteins were detected by Western blot analysis using antibodies against SMAD3 or SMAD4 (Cell Signaling) and KLF5.Wildtype FLAG‐SMAD3 and mutated SMAD3 (FLAG‐ mSmad3L for Smad3EPSM and FLAG‐mSMAD3C forSMAD3 3SA) plasmids, which were kindly provided by Dr. Kazuichi Okazaki of Kansai Medical University (Osaka, Japan),9 along with pcDNA3‐KLF5 andpcDNA3‐p300‐HA,19 were transfected into COS‐1 cellsusing the Lipofectamine reagent (Invitrogen). FLAG‐pcDNA3 vector served as a negative control.This assay was performed in HaCaT cells (5 ng/mL TGF‐ β for 3 hours in medium with 1% serum) with the oligonucleotides for p15 and Myc promoters (Inr and TIE)19,23 which were labeled with biotin on 5′‐end (MWG‐Biotech, High Point, NC). The procedures were previously described.19,23This assay was performed in HaCaT cells with Ras expression and TGF‐β treatment (5 ng/mL for 1 hour) by SimpleChIP Kit (Magnetic Beads; Cell Signaling Tech-nology). Antibody against KLF5 (Abcam Inc) was used to precipitate protein/DNA complex and the precipitated DNA was detected by real‐time PCR with specificprimers: 5′‐ATGCGTCCTAGCATCTTTGG‐3′ (p15 pro-moter forward); 5′‐TAGCGCGG‐ACGCAGCCGAGC‐3′ (p15 promoter reverse); 5′‐GGTCTGGACGGCTGAGGA CC‐3′ (Myc promoter forward); and 5′‐CTGCCTCTCG CTGGAATTAC‐3′ (Myc promoter reverse). Inr and TIE were covered by these two pair‐primers, respectively. The procedures followed the manufacturer’s protocol.All statistical analyses were conducted with SPSS 17.0 (SPSS Inc, Chicago, IL). Statistical differences among treatment groups and control were compared usingone‐way variance analysis. The Student’s t test (two‐ sided) was applied for comparisons involving two groups.
3|RESULTS
Previously, we investigated the function of TGF‐β and KLF5 in the regulation of cell proliferation and gene transcription using a well‐characterized cell line—the HaCaT human epidermal epithelial cell line19,20,23,30—in which we had established that TGF‐β‐induced KLF5 acetylation and the subsequent reassembly of the p15transcriptional complex to activate its transcription (Figure 1A). We used the same cell line for Ras experiments. We transfected HaCaT cells with retro-viruses expressing Ha‐RasG12V28 and established zeocin‐ resistant stable RasG12V‐expressing cell populations (HaCaT‐Ras) following the procedure described bySekimoto et al.9 As expected, Ras protein was expressed at much higher levels in HaCaT‐Ras cells when compared with control HaCaT cells (HaCaT‐zeocin)carrying empty vector (pBabe‐zeocin) (Figure 1B). In addition, 200‐nM siRNA specific for KLF5 (siKLF5) were transfected into HaCaT‐zeocin and HaCaT‐Ras cells for 48 hours to knock‐down KLF5 expression, and luciferase siRNA served as the negative control (Figure 1B). Cellswith different Ras and KLF5 status were then treated with 2 ng/mL of TGF‐β for 20 hours. With uninterrupted KLF5 expression, TGF‐β‐induced p15 expression and reduced Myc expression in HaCaT‐zeocin control cells (Figure 1B; lanes 1 and 2), but had hardly any effect on the expression of both p15 and Myc in HaCaT‐Ras cells in which the mutant RasG12V was expressed (Figure 1B;lanes 5 and 6), suggesting that Ras activation abolished the effect of TGF‐β on the expression of p15 and Myc.Contrarily, when KLF5 was knocked down, p15expression increased and Myc expression decreased (Figure 1B; lanes 1 and 3). Unexpectedly, Ras decreased the effect of KLF5 knockdown on p15 expression, although not obviously altering the effect of KLF5 knockdown on Myc expression (Figure 1B; lanes 3 and 7), which suggestedthe necessity of KLF5 for Ras to regulate p15 expression.
When both TGF‐β and Ras were present in the cells with reduced KLF5 expression, however, TGF‐β and Ras appeared to antagonize and negate each other’s effect; this led to the increased p15 and decreased Myc bythe knockdown of KLF5 (Figure 1B; lanes 1 and 8), an expression pattern similar to the pattern in cells with neither TGF‐β nor Ras (Figure 1B; lanes 1 and 3).At the messenger RNA (mRNA) level, as measured by real‐time PCR, the patterns of p15 and Myc expression in the context of KLF5 knockdown, TGF‐β treatment, and Ras expression were almost identical to the patterns oftheir protein expression, and the patterns only became more obvious (Figure 1C,D). For example, Ras activationsuppressed the effect of TGF‐β on the expression of p15 and Myc when KLF5 expression was maintained. Interestingly, knockdown of KLF5 reduced the effect ofRas on the function of TGF‐β, resulting in a certain level of restoration of TGF‐β‐regulated p15/Myc expression (Figure 1C,D). Moreover, we performed a cell cycledistribution test in HaCaT‐zeocin and HaCaT‐Ras cells transfected with SiKLF5 or control and treated with TGF‐ β through cell flow cytometric analysis, and found that both KLF5 knockdown and Ras activation abolished the cell cycle arrest function of TGF‐β, while KLF5 knock- down restored this function of TGF‐β to a certain level in HaCaT‐Ras cells (Figure S1). These results were con- sistent with previous findings that Ras suppressed the function of TGF‐β in the modulation of gene regulation and cell proliferation. In addition, whereas KLF5 is essential for TGF‐β to regulate gene expression in HaCaT cells, Ras activation altered the function of both KLF5 and TGF‐β in gene regulation.KLF5 acetylation is essential for TGF‐β to increase p15 expression and reduce Myc expression.19,20,23 In addition, acetylation reverses KLF5’s function in p15 transcriptionfrom a repressor to a transcription activator.19 Therefore, it is possible that Ras could modulate the function of TGF‐β in gene transcription via altering KLF5 acetyla-tion.
This was also suggested by discovering that Rasaltered the function of KLF5 in the regulation of p15 and Myc (Figures 1B and 1D). In testing this hypothesis, HaCaT‐zeocin and HaCaT‐Ras cells were treated with5 μm of trichostatin A for 4 hours, which enhanced thedetection of acetylation, and 5 ng/mL of TGF‐β for 1 hour. Western blot analysis showed that while TGF‐β‐ induced KLF5 acetylation in HaCaT‐zeocin cells as previously reported, it did not show such an effect in RasG12V‐expressing HaCaT‐Ras cells (Figure 1E and 1F). Interestingly, Ras alone increased the level of KLF5 acetylation, as did TGF‐β (Figure 1E; lanes 1, 3, 5, and 7). TGF‐β and Ras thus appeared to antagonize each other in the acetylation of KLF5 (Figure 1E,F). To confirm the effect of Ras on TGFβ‐inducing KLF5 acetylation, different combinations for FLAG‐tagged KLF5, p300, pcDNA3, Ha‐Ras, Ha‐RasG12V, and TGF‐β receptor I (TβRI) were transfected into HEK 293 cells, and acetylated KLF5 and total KLF5 were detected byimmunoprecipitation combined with Western blot ana- lysis. In these cells, the TGF‐β signal was provided by expressing the autoactivated TGF‐β type I receptor (TβRI), and the hyperactivated Ras signal was provided by the overexpression of RasG12V. We found that KLF5 acetylation was enhanced by p300 and TGFβ signaling, while both wildtype Ha‐Ras and mutated Ha‐Ras dramatically inhibited KLF5 acetylation (Figure 1G). These results indicated that TGF‐β could not induce the acetylation of KLF5 in the presence of active Rassignaling, which could be responsible for the inabilityof TGF‐β to induce p15 and repress Myc in RasG12V‐ expressing HaCaT cells.It was reported that hyperactive Ras changes SMAD2 and SMAD3 signaling through the phosphorylation of their linker regions,9 and both SMAD2 and SMAD3 arenecessary for TGF‐β and KLF5 to regulate gene expres- sion.19,20,23 To test whether Ras prevented TGF‐β‐induced KLF5 acetylation through the phosphorylation of SMAD2 and SMAD3, we treated serum‐deprived HaCaT‐zeocin and HaCaT‐Ras cells with 5 ng/mL of TGF‐β for 1 hour and measured the expression ofdifferent proteins. In HaCaT‐zeocin cells, TGF‐β induced the phosphorylation of SMAD3 at serine 214 (pSMAD3‐ S214) and threonine 179 (pSMAD3‐T179) (Figure 2A; four lanes at the left‐hand side).
The expression of RasG12V in HaCaT‐Ras cells, on the other hand, had little effect on the phosphorylation of S214 and T179 on itsown; however, enhanced TGF‐β‐induced phosphoryla- tion at these two sites (Figure 2A). TGF‐β alone had no effect on the phosphorylation of SMAD3 at serine204 (pSMAD3‐S204), while Ras dramatically increased the phosphorylation regardless of TGF‐β treatment (Figure 2A).In the CTR of SMAD3, where serines 423 and 425 (pSmad3‐C) were phosphorylated, TGF‐β alone induced a significant level of phosphorylation as expected, whileRas alone had no effect; however, it did suppress the effect of TGF‐β (Figure 2B).Although these results were consistent with previous findings that Ras increased the SMAD3 linker region phosphorylation (S214, T179, and S204) but inhibited SMAD3 CTR phosphorylation (S423/425), and that CTRphosphorylation was essential for TGF‐β and SMAD3 to activate growth‐inhibitory genes8; we wanted to know whether phosphorylation of SMAD3 modulated TGF‐β‐ induced KLF5 acetylation in the growth‐inhibitory function of TGF‐β; in addition, we wanted to determinewhether this modulation was interrupted by Ras. We transfected HaCaT cells with three different constructs of SMAD3, including the wildtype, a mutant (mSMAD3C) in which the three C‐terminal serine residues phosphory-lated by TβRI were mutated into alanines, and a mutantthat lacks four phosphorylation sites in the linker region (mSMAD3L). After transfection for 60 hours, cells were treated with TGF‐β (5 ng/mL for 1 hour). Western blotanalysis demonstrated that wildtype SMAD3 enhancedthe TGF‐β increased KLF5 acetylation, which was consistent with the previously established role of SMAD3in the acetylation of KLF5. Overexpression of mSMAD3L, which prevents linker region phosphorylation and enhances SMAD3 CTR phosphorylation, increased the level of KLF5 acetylation induced by TGF‐β (Figure 2C; lanes 2, 7, and 8).
Overexpression of mSMAD3C, whichprevents C‐terminal phosphorylation, interrupted the effect of TGF‐β on KLF5 acetylation (Figure 2C; lanes 4 and 6). Interestingly, we detected the expression of Myc and p15 mRNA in the above‐mentioned HaCaT cells transfected with wildtype SMAD3 and mutant SMAD3,and cells were treated with vehicle or TGF‐β at 2 ng/mL for 20 hours. We found that both the decrease of MycmRNA level and the increase of p15 mRNA level by TGF‐β in cells transfected with wildtype SMAD3 and mSMAD3L were significantly more than those in cellsoverexpressed in mSMAD3C (Figure 2D). These results suggested that SMAD3 CTR phosphorylation was neces- sary for TGF‐β to induce KLF5 acetylation, while SMAD3linker region phosphorylation played an opposite role.Because SMAD2 and SMAD3 are phosphorylated through similar mechanisms and exert similar functions, we also determined the effect of Ras signaling onthe phosphorylation of SMAD2. Expression of RasG12V‐enhanced TGF‐β‐induced SMAD2 linker regionphosphorylation (Figure 2E), but decreased TGF‐β‐ induced SMAD2 CTR phosphorylation (Figure 2F), which was similar to the results for SMAD3. Theseresults suggested that SMAD2, such as SMAD3, could regulate KLF5 acetylation.With the results that suggest Ras had a role in modulating TGF‐β function in gene regulation (Figure 1), we examined whether Ras modulated the TGF‐β‐induced p300‐KLF5‐SMADs transcriptional com- plex. We cotransfected p300, KLF5, and SMAD3 into COS‐1 cells and used p300 antibody to immunoprecipi-tate the protein complexes. While TGF‐β or Ras aloneclearly enhanced the binding of KLF5 to p300 without ectopic expression of SMAD3, the combination of TGF‐β and Ras completely interrupted the interaction betweenp300 and KLF5, as no KLF5 could be detected in the p300 precipitates (Figure 3A). This pattern was consistent with the pattern of KLF5 acetylation regulated by TGF‐β andRas signal (Figure 1E,F).To determine whether phosphorylation of SMAD3 affected the binding of KLF5 to p300, we transfected wildtype SMAD3, mutants mSMAD3L and mSMAD3C, KLF5, and HA‐tagged p300 into COS‐1 cells and used an antibody against HA tag to precipitate p300 complexes.We found that both SMAD3 and the linker region mutant mSMAD3L enhanced; however, the C‐terminal tailmutant mSMAD3C dramatically reduced the interaction between p300 and KLF5 (Figure 3B). In addition, while the linker region mutation did not affect the interactionbetween SMAD3 and p300, the CTR mutation obviously reduced the interaction (Figure 3B). These results indicated that the phosphorylation of SMAD3 at its CTR was necessary for the interactions among p300, SMAD3, and KLF5.We also used HaCaT cells expressing RasG12V to further examine whether Ras altered the function ofTGF‐β through impeding the p300‐KLF5‐SMADs tran- scriptional complex. Consistent with results from COS‐1 cells, the interaction between endogenous KLF5 and p300 was enhanced by either TGF‐β treatment or Rasexpression alone; however, the interaction was abolished by the combination of TGF‐β and Ras (Figure 3C; lanes 1‐ 4).
In addition, while the interaction between p300 andSMAD3 or that between p300 and SMAD4 was induced by TGF‐β treatment in HaCaT‐zeocin cells as expected, the expression of RasG12V abolished the interactions regardless of TGF‐β status, which was somewhatdifferent from the results in COS‐1 cells where SMAD3 was detected in Ras‐positive and TGF‐β negative cells. These results indicated that the TGF‐β‐induced interac-tions among p300, KLF5, SMAD3, and SMAD4 were interrupted by Ras activation in HaCaT cells.To further confirm this finding, we also used an antibody against SMAD4 to precipitate the protein complex from HaCaT cells and detected KLF5, p300, and SMAD3 by Western blot analysis. Interactions among p300, KLF5, SMAD3, and SMAD4 were againdetected in TGF‐β treated cells without Ras activation(Figure 3D; lanes 1 and 2). The expression of RasG12V, however, interrupted the interaction between SMAD4 and KLF5 or that between SMAD4 and p300; however, there were no obvious interaction effects between SMAD3 and SMAD4 (Figure 3D; lanes 2 and 4). These results further indicated that Ras activation interrupted the interaction of SMAD4 with KLF5 or p300, but not its interaction with SMAD3.Collectively, these results indicated that Ras activation suppressed the formation of TGF‐β‐induced p300‐KLF5‐ SMADs transcriptional complex formation, which couldlead to failures in KLF5 acetylation and transcriptional modulation of growth‐inhibitory genes.KLF5 is an important transcription factor that binds to the promoters of p15 and Myc in the absence and presence of TGF‐β.19,23 Because Ras suppresses TGF‐β‐induced KLF5 acetylation through impeding the forma-tion of the p300‐KLF5‐SMADs complex, but enhances KLF5 acetylation in the absence of TGF‐β and the p300‐ KLF5‐SMADs transcriptional complex, we investigatedwhether Ras affected the binding of KLF5 to the promoters of p15 and Myc by using the oligo pull‐down assays and chromatin immunoprecipitation (ChIP) as-says.
For the p15 promoter, oligonucleotides of the previously identified Inr site, which contained a con- sensus KLF5 binding CA box and could be bound only by acetylated KLF5,19 were incubated with cell lysates fromHaCaT‐zeocin and HaCaT‐Ras cells treated with 5 ng/mL TGF‐β or control solution for 1 hour. As expected, the binding of KLF5 to Inr was induced by TGF‐β in the absence of RasG12V expression (Figure 4A; lanes 1 and 2).In HaCaT‐Ras cells in which RasG12V was expressed, while Ras alone induced the binding of KLF5 to the Inr element, like TGF‐β, their combination abolished the binding (Figure 4A; lanes 3 and 4), indicating that Ras suppressed TGF‐β‐induced binding of KLF5 to the p15 promoter. For SMAD4, whose binding to the Inr element was also induced by TGF‐β and was detectable (Figure 4A; lanes 1 and 2), the expression of RasG12V prevented the binding regardless of TGF‐β status (Figure4A; lanes 3 and 3). Similarly, binding of KLF5 to the TGF‐ β responsive TIE element of the Myc promoter was enhanced by the TGF‐β treatment or RasG12V expression alone in HaCaT cells (Figure 4B; lanes 1 and 2); however,the expression of RasG12V abolished TGF‐β‐induced binding (Figure 4B; lanes 3 and 4). Moreover, throughthe ChIP assay, we confirmed the regulation of KLF5 binding on p15 promoter and Myc promoter by TGF‐β and RasG12V (Figure 4C). These results indicated that Ras suppressed TGF‐β‐induced binding of KLF5 to the promoters of p15 and Myc in HaCaT cells.
4| DISCUSSION
Previously, we reported that KLF5 was a part of the TGF‐ β‐induced SMADs complex, and that KLF5 acetylation was essential for TGF‐β to induce p15 and repress Myctranscription.19,20,23 KLF5 has also been implicated in the function of Ras signaling, as Ras activation induced KLF5 expression in epithelial cells and fibroblasts, and KLF5 alone could transform fibroblasts, similar to Ras, under certain conditions.11 Furthermore, Ras signaling com-promised the inhibitory effect of TGF‐β on cell prolifera-tion, and inhibition of Ras activities could restore TGF‐β’s growth‐inhibitory function.6 These studies im- plied that KLF5 could be the cross point between Ras and TGF‐β, and our findings from this study provided experimental evidence for this hypothesis. First, activa- tion of Ras signaling abolished the effects of TGF‐β on the induction of p15 and repression of Myc (Figure 1), whichdepended upon the acetylation of KLF5.19,23 Second, Ras signaling interrupted TGF‐β‐induced KLF5 acetylation(Figure 1), which was previously shown to be essential for the assembly of TGF‐β‐induced SMADs associated transcriptional complex.19,20,23 Ras activation, therefore, not only suppressed the formation of TGF‐β‐inducedp300‐KLF5‐SMADs transcriptional complex (Figure 3), but also prevented the TGF‐β‐induced binding of KLF5 onto the promoters of p15 and Myc (Figure 4A‐D). The effect of Ras activation on TGF‐β‐induced assembly of p300‐KLF5‐SMADs transcriptional complex onto thep15/Myc promoters was similar to that of the acetyla- tion‐deficient KLF5 mutant demonstrated in our previous studies,19,20,23 suggesting that the inhibition of KLF5acetylation by Ras was an important mechanism under- lying the inability of TGF‐β to properly regulate the expression of p15 and Myc in cells with activated Rassignaling.
Although Ras interrupted KLF5 acetylation, its inter- action with p300, and its binding to p15 and Myc promoters when both Ras signaling and TGF‐β signalingwere active, Ras signaling alone showed an oppositeeffect, which induced KLF5 acetylation and enhanced the interaction between p300 and KLF5 as well as the binding of KLF5 to p15 and Myc promoters (Figures 1, 3, and 4). Consistently, Ras activation alone increasedrather than decreased p15 expression (Figure 1B). Consistent with previous findings in which Ras activation induced senescence and cell cycle arrest via the induction of INK4 genes including p15, p16, and p19 in both fibroblasts and epithelial cells,31-33 these results suggestedthe involvement of KLF5 in Ras‐induced senescence andcell cycle arrest. Induction of senescence and cell cycle arrest are important mechanisms in the response of cellsto oncogenic signaling; therefore, the deletion of INK4 members abrogated Ras‐induced senescence and ren- dered cells susceptible to Ras‐mediated transforma-tion.31,33 It was demonstrated that the induction of INK4 genes was mediated by Raf/MEK/ERK signaling.34 Our earlier findings indicated that acetylated KLF5 wasessential for the proper assembly of transcription factor complex on the p15 promoter in TGF‐β‐induced expres- sion of p15.19,20 Therefore, it is possible that acetylatedKLF5 is also essential for Ras signaling to induce p15 and cause cell cycle arrest and senescence, and deletion or deacetylation of KLF5 could thus make cells susceptible to Ras‐mediated immortalization and transformation. We are currently testing these predictions.However, there were differences in the induction of p15 between Ras and TGF‐β signaling.
Although both induced the p300‐KLF5 interaction and the binding ofKLF5 to gene promoters (Figures 1, 3, and 4), TGF‐β‐ induced p300‐KLF5 complexes also contained SMADs (Figure 3 and Ref. [19,20]), but Ras‐induced p300‐KLF5 complexes did not involve SMADs (Figure 3C,D).Furthermore, the interaction between KLF5 and SMAD4 was interrupted by Ras activation (Figure 3D). Consis- tently, although TGF‐β induced the binding of both KLF5and SMADs to the p15 promoter, Ras only induced thebinding of KLF5, but not that of SMADs (Figure 4A). Therefore, KLF5 and p300 are needed by TGF‐β and Ras signaling in gene regulation; however, our study deter- mined that SMADs might only be needed by TGF‐β.Our previous studies demonstrated that SMAD3 interacts with KLF5 to mediate the function of TGF‐β in gene regulation.19,20 On the other hand, while bothTGF‐β and Ras signaling induced the phosphorylation of SMAD3, that induced by TGF‐β occurred at the CTR and was essential for TGF‐β’s inhibitory function, but that induced by Ras occurred at the linker region and had an antagonistic effect on TGF‐β’s inhibitory function.35,36 Collectively, we hypothesized a new mechanism for how Ras signaling interrupted the function of TGF‐β: Ras signaling interrupted TGF‐β‐induced KLF5 acetylation, which in turn altered the assembly of the p300‐KLF5‐SMADs complex onto gene promoters. Our findings in this study supported this hypothesis.In this study, we found that Ras oncogenic signaling interrupted TGF‐β‐induced KLF5 acetylation by inducingSMAD3 linker region phosphorylation and interfering with TGF‐β‐induced SMAD3 CTR phosphorylation. Subsequently, Ras activation interrupted the interactions between KLF5 and TGF‐β effectors, SMADs, and p300,and the binding of the KLF5‐SMADs complex to genepromoters. It has been reported that in keratinocytes, thrombin activates EGFR/ERK1/2 and subsequent phos- phorylation of the linker region of SMAD2 and induces the expression of PAI‐1.37 We, therefore, propose that during carcinogenesis, oncogenic signaling such as Ras signaling prevents the formation of TGF‐β‐inducedtranscriptional complexes containing acetylated KLF5and SMAD4; this leads to TGF‐β losing its antimitogenic and cell growth‐inhibitory functions, while phosphoryla- tion of the linker region of SMAD2 may result in the activation of other TGF‐β target genes, such as cell migration–related genes and epithelial‐mesenchymal transition transcription factors, and promotion oftumorigenesis.
In summary, we found that Ras activation in the absence of TGF‐β‐induced p300‐KLF5 interaction, KLF5 acetylation, and the binding of KLF5 on genepromoters to induce p15; however, in the presence of TGF‐β, Ras activation induced SMAD3/2 linker region phosphorylation, which suppressed the formation ofTGF‐β‐induced p300‐KLF5‐SMADs transcriptional complex, interrupted TGF‐β‐induced KLF5 acetyla- tion, prevented TGF‐β‐induced binding of the com- plex to the promoters of p15 and Myc, and abolished the functions of TGF‐β ML264 signaling on the transcription of p15 and Myc. These results implied that activationof Ras signaling regulated the formation of down- stream transcriptional complexes and target gene expression of the TGF‐β pathway by inducingSMAD3/2 linker region phosphorylation; it addition-ally supported that KLF5 acetylation was essential for the assembly of TGF‐β to induce transcriptional complex formation on the promoters of growth‐related target genes. Taken together, our findings suggest that interruption of KLF5‐SMADs complex formation by Ras activation is responsible for con- verting the function of TGF‐β from tumor suppressive to tumor promoting.