Tumour necrosis factor and PI3-kinase control oestrogen receptor alpha protein level and its transrepression function

Oestrogen receptor alpha (ERα) is an oestrogen-activated transcription factor, which regulates proliferation and differentiation of mammary epithelial cells by activating or repressing gene expression. ERα is a critical prognostic indicator and a therapeutic target for breast cancer. Patients with tumours that express higher level of ERα have better prognosis than patients with tumours that are ERα negative or express lower level of ERα. Better prognosis in ERα-positive patients is believed to be due to repression of proinvasive gene expression by ERα. Oestrogen receptor alpha represses gene expression by transrepressing the activity of the transcription factors such as nuclear factor-kappaB or by inducing the expression of transcriptional suppressors such as MTA3. In this report, we show that ERα transrepresses the expression of the proinvasive gene interleukin 6 (IL-6) in ERα-negative MDA-MB-231 breast cancer cells stably overexpressing ERα. Using these cells as well as ERα-positive MCF-7 and ZR-75-1 cells, we show that tumour necrosis factor alpha (TNFα) and the phosphatidylinositol-3-kinase (PI3-kinase) modulate transrepression function of ERα by reducing its stability. From these results, we propose that TNFα expression or PI3-kinase activation lead to reduced levels of ERα protein in cancer cells and corresponding loss of transrepression function and acquisition of an invasive phenotype.

Oestrogen receptor alpha (ERa) expression status is of prognostic significance for breast cancer. Breast cancer patients with the highest levels of ERa protein have a 90% 5-year survival rate and display very few p53 mutations. Patients with lower ERa levels have B45% 5-year survival and higher p53 mutation rates. The survival rate in these patients is similar to patients with a subset of ERa-negative breast cancer (Sorlie et al, 2001). Better prognosis in ERa-positive breast cancer patients can partly be attributed to their response to antihormone therapy (Ali and Coombes, 2002). However, because patients with higher rather than lower ERa protein have better prognosis, it is likely that some of the ERaregulated genes suppress invasion and metastasis of breast cancer. Consistent with this possibility, it was shown recently that ERadependent expression of metastasis associated protein 3 (MTA3) is required to prevent invasive growth of breast cancer cells (Fujita et al, 2003). Furthermore, a recent microarray study has shown that among B400 genes regulated by ERa/oestrogen in MCF-7 cells, majority of them (70%) are downregulated (Frasor et al, 2003). Some of the downregulated genes are known to be involved in invasion and homing of metastatic cancer cells (Muller et al, 2001).
Oestrogen receptor alpha is an oestrogen-activated transcription factor, which modulates gene expression by binding to oestrogen response elements (ERE) in the responsive promoter and through protein -protein interactions (Mangelsdorf et al, 1995;Di Croce et al, 1999). Oestrogen receptor alpha contains a central DNA binding domain (DBD), C-terminal ligand binding domain (LBD), as well as ligand-dependent activation function (AF-2) and Nterminal ligand-independent activation function (AF-1). Upon binding to oestrogen, ERa homodimers bind to ERE in the responsive gene promoters and activate gene expression. In addition, ERa homodimers activate non-ERE containing promoters by interacting with transcription factors such as SP-1 and AP-1 (Gaub et al, 1990;Paech et al, 1997;Dong et al, 1999). Transactivation by ERa involves ligand-dependent recruitment of coactivators, which serve as an intermediate between the receptor and the RNA polymerase II transcription complex (Horwitz et al, 1996;Glass and Rosenfeld, 2000). Although binding of oestrogen to LBD is essential for complete activation of ERa, phosphorylation by extracellular signal-activated kinases is thought to play a role in oestrogen-dependent and oestrogen-independent activity of ERa (Ali et al, 1993;Le Goff et al, 1994). Recently, a novel cell-type specific nongenomic action of ERa involving oestrogen-dependent association of ERa with phosphatidylinositol-3-kinase (PI3-kinase) leading to activation of the cell survival kinase AKT has also been reported (Simoncini et al, 2000). In addition, ERa localised in the plasma membrane has been shown to activate the MAP kinase pathway and contribute to growth regulation of breast cancer cells (Filardo et al, 2000;Marquez and Pietras, 2001;Razandi et al, 2003).
Transrepression of gene expression through protein -protein interaction is also a critical function of ERa. For example, inhibition of GATA-1-mediated transcription by ERa is responsible for suppression of erythroid differentiation by oestrogen (Blobel et al, 1995). The protective effect of oestrogen against sepsis is believed to be due to the suppression of proinflammatory gene expression (Schroder et al, 1998;Evans et al, 2002). Similarly, ERa-dependent repression of nuclear factor kappa B (NF-kB) activity is important for maintaining bone density (Jilka et al, 1992;Pottratz et al, 1994;Stein and Yang, 1995). Evans et al (2002) have identified several NF-kB-regulated genes that are repressed by ERa, which include antiapoptotic proteins GADD45b, apoptosis inhibitor 2, A20 and NF-kB p105 . A recent report suggested that ERa at higher levels reduces cancer cell growth and angiogenesis by inhibiting the expression of vascular endothelial growth factor . Furthermore, it was reported that unliganded and liganded ERa reduce cancer cell migration and invasion, through a mechanism that involves protein -protein interaction (Platet et al, 2000). Unlike the case of the glucocorticoid receptor (GR) where transrepression function is well characterised (Nissen and Yamamoto, 2000), the mechanism of ERa-mediated transrepression is not completely understood. Antagonism of NF-kB activity has been used as a model system to understand ERa-mediated transrepression. Repression of NF-kB activity by ERa is cell type specific (Cerillo et al, 1998). Both the DBD and LBD of ERa are essential for efficient repression of NF-kB activity (Stein and Yang, 1995;Valentine et al, 2000). It is suggested that ERa interacts directly with the Rel-homology domains (RHD) of the NF-kB subunits, p50 and p65, thereby interfering with the transcriptional activity of DNA-bound NF-kB (Stein and Yang, 1995). Apart from inhibition through direct protein -protein interaction, competition for the limiting amount of common coactivators such as SRC-1 and p300/CBP is suggested to play a role in ERa-dependent repression of NF-kB activity, although studies with ERa harbouring mutations in its transactivation domain fail to support such a mechanism (Sheppard et al, 1999;Harnish et al, 2000;Valentine et al, 2000).
The goals of this study were to determine whether ERa transrepresses the expression of interleukin 6 (IL-6), a cytokine that is linked to breast cancer cell invasion and motility as well as resistance to chemotherapy (Tamm et al, 1991;Conze et al, 2001), and to identify signalling pathways that may modulate transrepression by altering the stability of ERa. Using the ERa-negative breast cancer cell line MDA-MB-231 stably overexpressing ERa, we show that ERa transrepresses tumour necrosis factor alpha (TNFa)-inducible expression of IL-6. We also show that TNFa and PI3-kinase pathway modulate transrepression by reducing the stability of ERa.

MATERIALS AND METHODS
Generation of ERa-overexpressing cells MCF-7, ZR-75-1 and MDA-MB-231 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cDNA encoding ERa was cloned into the EcoRI site of the retroviral vector LxSN (pLxSN-ERa) (Miller and Rosman, 1989). Packaging of retrovirus and infection of MDA-MB-231 were performed as described previously (Newton et al, 1999). Briefly, AM12 cells were transfected with pLxSN or pLxSN-ERa expression vector and selected in media containing 600 mg ml À1 G418. G418resistant colonies were pooled and media supernatant with virus was used for infecting MDA-MB-231 cells. MDA-MB-231 cells were incubated with viral supernatant for 2 h in the presence of 8 mg ml À1 polybrene. The transduced cells were grown in the presence of G418 (1 mg ml À1 ). Individual G418-resistant colonies were isolated and ERa expression was measured by Western blotting. The constitutively active PI3-kinase expression vector (myr-PI 3-Kp110) was purchased from Upstate Biotechnology (Charlottesville, VA, USA). Constitutively active AKT (CA-AKT) and kinase-dead AKT (KD-AKT) have been described previously (Campbell et al, 2001). Cells were transfected with expression vectors using Lipofectamine 2000 transfection reagent as recommended by the manufacturer and analysed for ERa protein levels 48 h after transfection (Invitrogen, Carlsbad, CA, USA).

Northern blot analysis
Total RNA was prepared using the RNAeasy kit from Qiagen (Valencia, CA, USA). RNA was subjected to Northern blot analysis as previously described (Newton et al, 1999). Interleukin-6 cDNA was purchased from ATCC, whereas tomour necrosis factor receptor associated protein 1 (TRAF-1) cDNA has been described previously (Rothe et al, 1994).

ERa reduces TNFa-inducible IL-6 but not TRAF-1 expression
Previously, we reported constitutive NF-kB activation in the ERanegative breast cancer cell line MDA-MB-231, which correlated with increased expression of several NF-kB-inducible genes including IL-6, Mn-SOD, cIAP-2 and TRAF-1 (Patel et al, 2000). In addition, using transient transfection assays, we showed transrepression of NF-kB activity by ERa in these cells (Nakshatri et al, 1997). To further characterise the transrepression function of ERa, we stably overexpressed ERa in MDA-MB-231 cells using retrovirus-mediated gene transfer ( Figure 1A). Oestrogen receptor alpha-3 and ERa-6 clones overexpress wild-type ERa, whereas ERa-8 and ERa-9 overexpress mutant ERa (C530R), which cannot activate transcription of an ERE-containing reporter gene (data not shown). Cysteine 530 is within the recently identified KCK motif involved in intramolecular AF-1 and AF-2 interaction and this mutation reduces the affinity of ERa to oestrogen (E2) (Metivier et al, 2002). Cells expressing the mutant protein were used to evaluate transrepression independent of coactivator competition and by a mutant ERa with reduced affinity to E2. We compared the TNFa-inducible expression of NF-kB-regulated genes IL-6 and TRAF-1 in parental and ERa-overexpressing cells. Inducible IL-6 but not TRAF-1 expression was lower in ERa-3 and ERa-6 cells compared to LxSN2 cells ( Figure 1B). Similarly, IL-6 expression was lower in ERa-8 cells compared to LxSN23 cells ( Figure 1B). Tumour necrosis factor alpha (TNFa)-inducible expression of cIAP-2, another NF-kB regulated gene, was not influenced by ERa suggesting that transrepression is promoter-context dependent (data not shown). Transrepression of Mn-SOD expression by ERa was observed with early passage cells but not in late passage cells, which suggests existence of inherent mechanism to overcome transrepression (data not shown). As mutant ERa(C530R) transrepressed IL-6 expression, sequestration of common limiting coactivators by ligand-activated ERa is not necessary for transrepression.

Pure antioestrogen ICI182780 reverses transrepression function of ERa
To further confirm the role of ERa in reducing TNFa-inducible expression of IL-6 in ERa-overexpressing cells, we preincubated cells with E2, tamoxifen or ICI182780 for 2 h and measured IL-6 expression with or without TNFa treatment for 16 h. Binding of E2 leads to activation and subsequent proteosome-dependent degradation of ERa (Lonard et al, 2000). Previous studies have shown that binding of tamoxifen to ERa leads to stabilisation, whereas binding to ICI182780 leads to degradation of ERa without activation (Ali et al, 1993). Tamoxifen stabilised, whereas E2 and ICI182780 reduced ERa level in ERa-6 cells ( Figure 2A). The effect of various ligands on basal and TNFa-inducible IL-6 and TRAF-1 expression was examined. Oestrogen reduced basal IL-6 expression in ERa-6 cells compared to LxSN2 cells ( Figure 2B, middle panel). This could be due to sequestration and subsequent degradation of common coactivators by ERa. Interestingly, TNFa-inducible IL-6 expression was not influenced by E2. TNFa-induced IL-6 expres-sion was still lower in tamoxifen-treated ERa-6 cells compared to tamoxifen-treated LxSN2 cells, suggesting that tamoxifen-bound ERa is capable of transrepression, consistent with our previous transient transfection studies (Nakshatri et al, 1997). In contrast, TNFa-inducible IL-6 expression was similar in ICI182780-treated ERa-6 and LxSN2 cells ( Figure 2B, compare lanes 8 and 16). Note that none of the ligands altered TNFa-inducible TRAF-1 expression, which suggests that the observed effect of ligands on IL-6 expression is not due to toxicity. From these results, we conclude that ERa is responsible for lower IL-6 expression in ERa-6 cells compared to LxSN2 cells, and antioestrogens that destabilize ERa can overcome ERa-mediated suppression of IL-6 expression.

TNFa reduces the stability of ERa protein
We consistently observed a lower level of ERa in TNFa-treated cells compared to untreated cells and a further enhancement of ICI182780-dependent degradation of ERa by TNFa (Figure 2A). In early passage cells, TNFa reduced ERa protein level by as much as 60% (data not shown). This raised the possibility that TNFa modulates transrepression function of ERa by inducing its degradation. Towards this end, ERa-6 and ERa-8 cells with or without prior treatment with TNFa for 16 h were incubated with cyclohexamide to block protein synthesis. Cells were harvested at specific time intervals and ERa protein was measured by Western blotting. Oestrogen receptor alpha stability was much lower in cells pretreated with TNFa compared to untreated cells ( Figure 3A). Oestrogen receptor alpha undergoes proteosome-mediated degradation in TNFa-treated cells as the proteosomal inhibitor MG132 prevented ERa degradation ( Figure 3B). Neither caspase inhibitors nor calpain inhibitors altered the stability of ERa under untreated and TNFa-treated conditions (data not shown). Oestrogen receptor ER ER ER -6 ER -3 ER -8 ER -9 LxSN2 LxSN23 Oestrogen receptor alpha expression in cells transduced with retrovirus without the ERa coding sequence (LxSN2 and LxSN23) or with the ERa coding sequence (ERa-3, ERa-6, ERa-8 and ERa-9) was measured by Western blotting. Note that ERa-3 and ERa-6 cells express the wild-type receptor, whereas ERa-8 and ERa-9 cells express mutant receptor. (B) TNFa-inducible IL-6 but not TRAF-1 expression is lower in ERa-overexpressing cells compared to control cells. Cells were treated with TNFa for the indicated times and IL-6 or TRAF-1 expression was measured by Northern blot analysis. The same blot was reprobed with ribosomal protein gene 36B4 to ensure equal loading.  Tam  ICI  E2  Tam  ICI  _  E2  Tam  ICI  _  E2  Tam  ICI   +TNF  +TNF   _  _  E2  Tam  ICI  E2  Tam  ICI  alpha transcript levels were similar in untreated and TNFa-treated cells, suggesting that the effect of TNFa on ERa is at the level of protein stability ( Figure 3C). None of the effects of TNFa on ERa is due to TNFa-induced apoptosis of MDA-MB-231 as these cells were resistant to TNFa irrespective of ERa overexpression (data not shown). Tumour necrosis factor alpha-induced destabilisation of ERa was not restricted to MDA-MB-231 cells as TNFa reduced ERa protein in ERa-positive MCF-7 and ZR-75 cells ( Figure 3D). Consequences of ERa degradation on transrepression in MCF-7 cells could not be studied because of lack of IL-6 expression in these cells and their sensitivity to TNFa-induced apoptosis (data not shown).

Phosphatidylinositol-3-kinase inhibitor LY294002 stabilises ERa protein in MDA-MB-231 cells and inhibits TNFa-induced but not E2-induced degradation of ERa in MCF-7 cells
MAPK, cyclin A/cdk2, AKT, RSK2, PKA, PAK1, p38 kinase and Src phosphorylate ERa (Ali et al, 1993;Lee and Bai, 2002;Wang et al, 2002). Phosphorylation leads to ligand-independent activation in most cases, and activated ERa undergoes coactivator-ubiquitindependent degradation (Lonard et al, 2000). Phophatidylinositol-3-kinase, which is upstream of AKT, as well as MAPK are constitutively active in MDA-MB-231 cells and may promote phosphorylation-dependent degradation of ERa (Ma et al, 2001;Sliva et al, 2002). Consistent with this possibility, ERa showed ligand-independent activity in ERa-3 and ERa-6 cells (data not shown). To investigate whether any of these kinases determine the stability of ERa and thus modulate transrepression function, we treated cells with various inhibitors for 16 h with or without TNFa treatment and measured ERa protein. The MAP kinase inhibitor PD98059, PKA inhibitory peptide or Src kinase inhibitor did not alter ERa protein level in ERa-6 cells ( Figure 4A). In contrast, the PI3-kinase inhibitor LY294002 stabilised ERa protein under both untreated and TNFa-treated conditions. Similar results were obtained in ERa-8 cells. Increase in ERa protein in LY294002treated cells was not due to increased transcription of ERa in LY294002-treated cells ( Figure 4B). To further confirm the role of PI3-kinase on ERa stability, we examined the effect of LY294002 on TNFa-induced degradation of ERa in MCF-7 cells. Although LY294002 reduced the basal ERa protein level possibly due to its effects on general transcription, it blocked TNFa-induced but not E2-induced degradation of ERa ( Figure 4C). Recent studies have shown that LY294002 inhibits both PI3-kinase and casein kinase II (Davies et al, 2000). We used apigenin, a casein kinase II inhibitor (Critchfield et al, 1997), to support our conclusion that PI3-kinase is involved in TNFa-induced degradation of ERa. Apigenin failed to inhibit TNFa-induced degradation of ERa in MCF-7 cells ( Figure 4D). In fact, apigenin on its own reduced ERa level. To further confirm the role of PI3-kinase in destabilisation of ERa, we transfected MCF-7 cells with constitutively active PI3-kinase and measured ERa protein 48 h after transfection. Oestrogen receptor alpha protein levels were lower in cells transfected with PI3-kinase expression vector ( Figure 4E). Similar results were obtained in 293 and MDA-MB-231 cells transfected with ERa and constitutively active PI3-kinase (data not shown). Activation of AKT alone is sufficient for PI3-kinase-mediated destabilisation of ERa as a constitutively active AKT (CA-AKT) but not kinase-dead AKT (KD-AKT) reduced ERa levels ( Figure 4E). It is possible that PI3kinase-mediated destabilisation of ERa involves AKT-dependent phosphorylation of ERa followed by activation-coupled degradation. Our repeated attempts to establish MDA-MB-231 cells overexpressing ERa mutants that cannot be phosphorylated by cyclin A/cdk2 (S102N,104P,106A), MAPK/cdk7 (S118A), AKT/RSK (S167A), PKA (S236A) or SRC (Y537F) were not successful. In transient transfection assays, phosphorylation-defective mutants were always expressed at a higher level than wild-type ERa (although expressed from a same promoter), suggesting that phosphorylation-defective mutants are more stable than wild-type ERa (data not shown).

Prolonged exposure of ERa-overexpressing cells to TNFa leads to loss of transrepression, which can be reversed partially by LY294002
To determine the consequences of stabilisation of ERa by LY294002 on transrepression, we treated parental and ERaoverexpressing cells with TNFa for 16 h or 3 days and measured IL-6 expression levels. Interleukin-6 expression in ERa-8 cells was lower than in parental cells after 16 h of TNFa treatment ( Figure 5). However, IL-6 expression was similar in both LxSN23 and ERa-8 cells after 3 days of TNFa treatment. Interestingly, LY294002 was more effective in reducing TNFa-inducible IL-6 expression in ERa-8 cells compared to LxSN23 cells. Similar results were obtained in ERa-6 cells. Note that LY294002 had no effect on TNFa-inducible expression of TRAF-1. Thus, inhibition of TNFa-inducible IL-6 expression by LY294002 is less likely due to reduction in AKT/ PKB-mediated activation of NF-kB or toxicity. We propose that LY294002 reduces TNFa-inducible IL-6 expression in ERa-overexpressing cells by enhancing transrepression function of ERa. In contrast to enhanced transrepression, LY294002 reduced transactivation by ERa (data not shown, Kishimoto and Nakshatri, submitted). Surprising specificity of LY294002 in inhibiting IL-6 but not TRAF-1 expression is encouraging as inhibitors with similar properties can be used to reduce invasion of breast cancer cells, more so of ERa-positive cancer cells, by specifically reducing IL-6 expression.

DISCUSSION
In this report, we show that the ERa protein level in breast cancer cells is regulated by TNFa and PI3-kinase, which has important implications on the transrepression function of ERa. Transrepression by ERa is believed to be responsible for reducing invasion and metastasis of ERa-positive breast cancers (Platet et al, 2000). Repression of gene expression appears to be a major function of ERa as recent studies show that among B400 genes regulated by ERa in MCF-7 cells, B70% of them are downregulated (Frasor et al, 2003). By lowering the ERa protein level, TNFa and PI3kinase can overcome transrepression by ERa, thus promoting invasion and metastasis of breast cancers. Recent molecular profiling data with patient samples is consistent with the above observation. Patients with lower levels of ERa protein in their tumours have shorter disease-free survival rates than patients with higher levels of ERa in their tumours (Sorlie et al, 2001). It is interesting that PI3-kinase levels are higher in highly invasive and metastatic breast cancer cell line MDA-MB-231 cells compared to nonmetastatic MCF-7 cells (Sliva et al, 2002), which can explain for LY294002-induced stabilisation of ERa in MDA-MB-231 cells overexpressing ERa. Although our efforts to generate MCF-7 and MDA-MB-231 cells lacking PI3-kinase using siRNA technique were not successful, the failure of other kinase inhibitors including PD98059, PP2 and apigenin to alter the stability of ERa suggests that specific inhibition of PI3-kinase is responsible for LY294002induced stabilisation of ERa. The PI3-kinase pathway appears to be involved in destabilisation of ERa by TNFa but not E2, as LY294002 inhibited TNFa-induced but not E2-induced degradation of ERa in MCF-7 cells (Figure 4). Our results differ to some extent from a recent report, which showed enhanced turnover of unliganded and liganded ERa in MCF-7 cells treated overnight with LY294002 (Marsaud et al, 2003). Authors suggested that PI3kinase activity is required for stabilisation of ERa in MCF-7 cells. Some of the effect of LY294002 on ERa protein level in MCF-7 cells could be at the level of ERa transcription from the endogenous promoter as we also observed similar decrease in ERa protein in MCF-7 cells treated with LY294002. However, this is not the case when ERa is expressed through a heterologous promoter as LY294002 stabilised ERa in MDA-MB-231 derivatives ERa-6 and ERa-8 cells ( Figure 4A). Several recent studies suggest that data generated using LY294002 alone should be interpreted cautiously. For example, LY294002 inhibits both PI3-kinase and casein kinase II at same concentration (Davies et al, 2000). LY294002 has been shown to bind to the LBD of ERa and inhibit its activity (Pasapera Limon et al, 2003). To ensure a direct role for PI3-kinase in destabilisation of ERa, we determined the effect of overexpression of constitutively active PI3-kinase on ERa protein levels in MCF-7 cells. PI3-kinase reduced ERa protein level in these cells ( Figure 4E).  Figure 5 LY294002 inhibits TNFa-induced IL-6 but not TRAF-1 expression. Cells were treated with TNFa for 16 h or 3 days with or without LY294002. The media was changed daily with the addition of fresh TNFa and LY294002. Interleukin-6 and TRAF-1 expression levels were measured by Northern analysis.
A major implication of this study is on sensitivity of breast cancer cells to chemotherapy. Interleukin-6 has been shown to increase motility and confer multidrug resistance to breast cancer cells (Tamm et al, 1991;Conze et al, 2001). By reducing basal and/or TNFa-induced IL-6 expression, ERa can reduce multidrug-resistant growth of breast cancer cells. Consistent with this possibility, in preliminary studies, we have observed increased sensitivity of ERa-overexpressing MDA-MB-231 cells to doxorubicin (data not shown). PI3-kinase inhibitors may further enhance the sensitivity of ERa-expressing cells to chemotherapy by stabilising ERa. We have recently observed inhibition of the transactivation function of ERa in tamoxifen-resistant breast cancer cells by PI3-kinase inhibitors (Kishimoto and Nakshatri, submitted). From these results, we propose that PI3-kinase inhibitors have the potential to overcome the multidrug resistance of ERa-positive breast cancers by simultaneously increasing transrepression and reducing transactivation by ERa.
How TNFa and PI3-kinase promote degradation of ERa remains to be determined. Although TNF-induced degradation of ERa has been reported, to our knowledge, this is the first report demonstrating a role for PI3-kinase in ERa degradation (Danforth and Sgagias, 1993). Phosphatidylinositol-3-kinase has recently been shown to be required for degradation of b-arrestin-1 in response to chronic insulin treatment (Dalle et al, 2002). It is possible that TNFa and PI3-kinase induces the expression of a protein that targets ERa for proteosome-mediated degradation or it may induce the activity of proteosomal subunits, which alters the specificity of the proteosome. In this regard, it has been shown that TNFa increases ubiquitin-conjugating activity by increasing the expression of UbcH2 through NF-kB (Li et al, 2003). The other possibility is that TNFa alters the NEDD8 pathway, which has recently been shown to be involved in ERa degradation (Fan et al, 2003). Peroxisome proliferator-activated receptor gamma (PPARg) and aryl hydrocarbon receptor also promote proteosomal degradation of ERa (Qin et al, 2003;Wormke et al, 2003). It is possible that TNFa utilises these receptors to promote ERa degradation. In this regard, kinetics and degree of TNF and aryl hydrocarbon receptor-induced degradation of ERa are similar. The other possibility is that TNFa, like IFNa, induces the replacement of proteosomal subunits, resulting in altered proteolytic specificity (Hisamatsu et al, 1996).
Although interaction between NF-kB and ERa was reported about 8 years ago (Stein and Yang, 1995), how that interaction leads to either transactivation or transrepression is not known. Initial studies suggested that competition for limiting coactivators is responsible for transrepression. However, subsequent studies by Parker's group and this study with a mutant ERa rule out coactivator competition as being the primary mechanism of transrepression, at least in breast cancer cells (Sheppard et al, 1999;Harnish et al, 2000;Valentine et al, 2000). Also, our studies show that not all NF-kB-regulated genes are transrepressed by ERa, which suggests that transrepression involves specific promoter context. There may be similarity in transrepression by GR and ERa. Expression of IL-8 upon TNFa stimulation involves NF-kB-dependent assembly of the transcription preinitiation complex followed by phosphorylation of the RNA polymerase II carboxyl terminal domain. Glucocorticoid receptor has been shown to interfere with phosphorylation of the RNA polymerase II carboxyl terminal domain without interfering with the preinitiation complex formation (Nissen and Yamamoto, 2000). Transrepression by GR in some instances involves corecruitment of the coactivator molecule GRIP1, and the coactivators SRC-1 and SRC-3 cannot substitute this function of GRIP1 (Rogatsky et al, 2002). Promoter specificity in ERa-mediated transrepression may also involve a similar mechanism.