Because phosphorylation is an important step in the activation and function of IRFs, we employed the natural kinase inhibitor apigenin to evaluate its effects on IRF6 phosphorylation. Surprisingly, treatment of the mammary epithelial cell line MCF-10A with apigenin resulted in an increase in IRF6 phosphorylation (denoted by the upper band of a doublet in Western blotting) (2
), suggesting an alternate mechanism apart from apigenin's kinase-inhibitory properties (Fig. ). Importantly, maspin expression appeared unchanged. As an internal control, a decrease in phospho-specific Akt levels verified apigenin activity.
FIG. 1. Proteasomal inhibition induces IRF6 phosphorylation. (A) Western blot analysis of MCF-10A cells treated with two different concentrations of apigenin (api) for 18 h. The IRF6 doublet represents phosphorylated (upper band, denoted by “p”) (more ...)
Based on apigenin's reported proteasome inhibitory properties, we tested whether the increase in IRF6 phosphorylation could be replicated following proteasomal inhibition. MCF-10A cells were treated with two well-characterized proteasome inhibitors (MG-132 and epoxomicin) for 18 h, and IRF6 phosphorylation was assayed by Western blotting (Fig. ). Similar to the effects of apigenin, proteasomal inhibition resulted in the phosphorylation of nearly the entire cellular pool of IRF6, whereas maspin again appeared to be unaffected by the treatment. Densitometric analysis further revealed a moderate increase in total IRF6 protein levels following proteasomal inhibition. Notably, an increase in IRF6 phosphorylation was not observed following treatment with chloroquine, a lysosomotropic agent which disrupts normal lysosomal function, despite a similar increase in IRF6 protein levels. Proteasomal inhibition induced a similar effect on IRF6 in primary human mammary epithelial cells (HMEpC) and the normal prostate cell line MLC8891, demonstrating that these effects are not cell type or tissue specific (Fig. ). The increase in IRF6 protein levels induced by MG-132 was confirmed by immunofluorescence microscopy, which also demonstrates the cytoplasmic localization of phosphorylated IRF6 following proteasomal inhibition (Fig. ).
The proteasome plays a critical role in cell cycle regulation, and therefore, we tested whether cellular proliferation altered the phosphorylation status of IRF6. Following the induction of cell cycle arrest in MCF-10A cells by serum deprivation, pharmacological cell cycle inhibition, or cell contact-dependent cell cycle arrest, the status of IRF6 phosphorylation was assessed by Western blotting (Fig. ). Interestingly, quiescent cells predominantly expressed nonphosphorylated IRF6. Furthermore, total IRF6 protein levels were notably increased in arrested cells compared to their levels in asynchronous cells in growth phase (50% confluent), indicating that cell proliferation directly regulates IRF6 protein expression and phosphorylation and suggesting that the phosphorylation of IRF6 may lead to decreased protein stability and/or targeted protein degradation. We employed real-time PCR to ascertain whether increased IRF6 mRNA expression might account for a portion of the total increase in IRF6 protein levels (Fig. ). Importantly, only the serum-starved cells demonstrated a substantial increase in IRF6 mRNA, suggesting that the increase in IRF6 protein in confluent or MG-132-treated cells results from increased protein stabilization and/or a decrease in targeted degradation. The increase in IRF6 protein levels was verified by immunofluorescence microscopy (Fig. ).
FIG. 2. Cell cycle arrest causes an increase in IRF6 protein levels and a reduction in IRF6 phosphorylation. (A) Western blot analysis of MCF-10A cells comparing proliferating cells (growth phase; 50% confluent) to quiescent cells arrested by serum starvation (more ...)
We next evaluated the relative stability of the IRF6 protein in proliferating versus quiescent MCF-10A cells using a pulse-chase assay. Our results demonstrate a substantial reduction of radiolabeled IRF6 in proliferating cells following a 30-min chase, whereas radiolabeled IRF6 remains detectable following a 6-h chase in confluent cells (Fig. ). Notably, the upper (phosphorylated) IRF6 band appears to diminish more quickly than the lower band, suggesting an increased stability of nonphosphorylated IRF6.
Since IRF6 expression and phosphorylation appear to be regulated by cell proliferation, we next investigated at what stage of the cell cycle IRF6 becomes phosphorylated. MCF-10A cells were synchronized by serum deprivation, and IRF6 phosphorylation was analyzed at multiple time points following the readdition of serum. The results shown in Fig. demonstrate that IRF6 phosphorylation is an early event in cell cycle progression, with maximal IRF6 phosphorylation occurring 30 min to 1 h following the addition of serum. Importantly, by 2 h, total IRF6 protein levels appeared to steadily decline, as determined by densitometric analysis. This was achieved primarily through a decrease in phosphorylated IRF6 levels, whereas the level of nonphosphorylated IRF6 appeared to change very little following the initial decrease coincident with serum addition. Importantly, the decrease in IRF6 expression occurred concomitantly with the appearance of cyclin D3, an important marker of the G1 phase of the cell cycle. Maspin levels were not significantly altered over the 18 h following the serum addition. These data support the possibility that IRF6 phosphorylation may facilitate its degradation, which in turn may allow progression from G0 (quiescence) to the G1 phase of the cell cycle.
FIG. 3. IRF6 protein expression and phosphorylation are regulated by the cell cycle in a proteasome-dependent manner. (A) Western blot analysis depicting changes in IRF6 phosphorylation and expression upon addition of serum to synchronized MCF-10A cells. Time (more ...)
To test whether IRF6 phosphorylation results in protein degradation via a proteasome-dependent pathway, serum was added to synchronized MCF-10A cells in the presence or absence of the proteasome inhibitor MG-132. Consistent with previous results, IRF6 protein levels noticeably decreased in the absence of MG-132 after 6 h following serum addition. However, in the presence of MG-132, total IRF6 protein levels were unchanged over 6 h, even though IRF6 was phosphorylated (Fig. ). Interestingly, in the absence of serum, MG-132 retained its ability to induce IRF6 phosphorylation. The exact mechanism for the phosphorylation of IRF6 following proteasomal inhibition in quiescent cells is not known.
Polyubiquitination of the target protein is the most common signal for proteasome-mediated degradation. To test whether IRF6 is ubiquitinated following phosphorylation, we performed coimmunoprecipitation analysis with an antiubiquitin antibody, in which we compared serum-starved cells (nonphosphorylated IRF6) with serum-stimulated cells (phosphorylated IRF6). Figure demonstrates that ubiquitin conjugation specifically occurs on phosphorylated IRF6 in serum-stimulated cells, which supports the hypothesis that the phosphorylation of IRF6 is a signal for proteasome-mediated degradation.
We next sought to identify the serum component responsible for IRF6 phosphorylation in MCF-10A cells. Because IRF6 phosphorylation occurs quickly after serum stimulation, we tested whether the mitogenic signaling of EGF, insulin, or hydrocortisone affected IRF6 phosphorylation. When added in concentrations similar to those present in complete growth medium, these growth factors were not sufficient to induce rapid IRF6 phosphorylation (Fig. ). Furthermore, neither the serum supplement MitoPlus, which contains an assortment of growth factors and hormones, nor charcoal-dextran-stripped serum were able to efficiently induce IRF6 phosphorylation. Expectedly, maspin levels remained constant. We are currently working to identify the unknown serum component(s) responsible for the induction of IRF6 phosphorylation.
Mammary gland development and differentiation are characterized by heightened periods of proliferation (during pregnancy) prior to the functional differentiation of the secretory lobuloalveolar cells which comprise the lactating gland. We therefore sought to confirm our findings in situ using immunohistochemistry to compare IRF6 expression levels during pregnancy and lactation in mice. The results shown in Fig. demonstrate a noticeable increase in IRF6 immunoreactivity in the lactating lobuloalveolar cells compared to the IRF6 immunoreactivity in ductal and glandular epithelial cells during pregnancy. A mild increase was also observed in maspin expression, an observation which coincides with the findings in a previous report showing maximal expression of maspin mRNA during lactation in the differentiating gland (34
). Quantification of the relative mean intensity of the staining indicated a greater-than-twofold increase in IRF6 immunoreactivity, which is very similar to the increase observed as MCF-10A cells become confluent in culture (Fig. and Fig. , respectively).
FIG. 4. IRF6 expression is maximal during lobuloalveolar differentiation (lactation). (A) Immunohistochemistry demonstrating increased IRF6 immunoreactivity during lactation compared to immunoreactivity during pregnancy. Wild-type C57/Black6 mice were harvested (more ...)
These findings led us to postulate that IRF6 is an important component of cell cycle regulation. Hence, we evaluated the effects of IRF6 reexpression on cell proliferation in breast cancer cells which no longer express readily detectable amounts of IRF6. Proliferation assays comparing IRF6-transfected breast cancer cells with control cells revealed a significant reduction in total cell number(s) following IRF6 transfection (Fig. ). In both poorly aggressive MCF-7 and highly aggressive MDA-MB-231 breast cancer cells, the reexpression of IRF6 reduced cellular proliferation by more than 40%. Furthermore, the reexpression of maspin in combination with IRF6 synergistically augmented the growth-inhibitory effects of IRF6, with the most-pronounced effect occurring in the highly aggressive cells, where the total cell number was reduced by nearly 90%. Importantly, MCF-7 and MDA-MB-231 cells stably transfected with maspin alone do not exhibit significantly altered doubling times. Real-time PCR analysis of IRF6-transfected MCF-7 cells indicated an 80% reduction in the cell proliferation marker Ki-67, suggesting an important role for IRF6 in promoting cell cycle arrest (Fig. ).
FIG. 5. IRF6 reexpression promotes cell cycle arrest in cancer cells. (A) Proliferation assay of poorly invasive (MCF-7) or highly invasive (MDA-MB-231) breast cancer cells infected with IRF6-expressing adenovirus (Ad IRF6) in the presence or absence of maspin, (more ...)