IFN-γ-induced expression of MUC4 in pancreatic cancer cells is mediated by STAT-1 upregulation: a novel mechanism for IFN-γ response

Abstract

MUC4 is a transmembrane mucin, which is aberrantly expressed in pancreatic adenocarcinoma with no detectable expression in the normal pancreas. Here, we present a novel mechanism of IFN-γ-induced expression of MUC4 in pancreatic cancer cells. Our studies highlight the upregulation of STAT-1 as a basis for MUC4 induction and demonstrate that its activation and upregulation by IFN-γ are two distinct, albeit temporally integrated, signalling events that drive the selective induction of IRF-1 and MUC4, respectively, within a single cell system. The profile of interferon regulatory factor (IRF)-1 gene induction by IFN-γ is consistent with its rapid transactivation by phospho-Y701-STAT-1. In contrast, the induction of the MUC4 mucin gene expression is relatively delayed, and occurs only in response to an increase in STAT-1 expression. A progressive binding of STAT-1 to various γ-interferon-activated sequences (GAS) in the MUC4 promoter is observed in chromatin immunoprecipitation assay, indicating its direct association. Stimulation of STAT-1 expression by double-stranded polynucleotides or ectopic expression is shown to induce MUC4 expression, without Y701 phosphorylation of STAT-1. This effect is abrogated by short interfering RNA (siRNA)-mediated inhibition of STAT-1 expression, supporting further the relevance of STAT-1 in MUC4 regulation. In conclusion, our findings identify a novel mechanism for MUC4 regulation in pancreatic cancer cells and unfold new perspectives on the foundation of IFN-γ-dependent gene regulation.

Introduction

MUC4 is aberrantly expressed in the majority (70–80%) of pancreatic tumours and tumour cell lines, while remaining undetectable in the normal pancreas (Andrianifahanana et al., 2001). Recent studies have provided compelling evidence implicating the MUC4 mucin in the pathogenesis of pancreatic cancer (Singh et al., 2007). Comparative expression analyses have revealed a positive correlation between MUC4 levels, the differentiation status of pancreatic tumour cell lines (Andrianifahanana et al., 2001) and tumour grading (Swartz et al., 2002). Moreover, functional studies on MUC4 have provided substantial evidence for its role in the promotion of pancreatic tumour cell growth and metastasis (Singh et al., 2004). Taken together, these observations suggest an intimate link between aberrant MUC4 expression and the pathogenesis of pancreatic cancer. Therefore, an improved understanding of the mechanism(s) underlying the regulation of this mucin gene may help in identifying key biochemical events and biologically relevant factors that may account for its aberrant upregulation in pancreatic tumour cells.

IFN-γ is a pro-inflammatory cytokine, which is secreted by activated T lymphocytes and natural killer cells and regulates a variety of physiological responses (Kalvakolanu and Borden, 1996). The binding of IFN-γ to its cell surface receptor activates the receptor-associated tyrosine kinases JAK1 and JAK2. JAKs phosphorylate and activate the latent cytosolic transcription factor STAT-1, which then dimerizes, translocates to the nucleus and binds to the γ-interferon-activated sequence (GAS) elements of IFN-γ-responsive genes, resulting in gene activation (Darnell et al., 1994). STAT-1 is a remarkably versatile transcription factor that regulates gene expression in multiple forms. The role of Y701-phosphorylated STAT-1 in inducing immediate-early response genes is well established (Darnell et al., 1994). Likewise, its phosphorylation at S727 alone is fundamental to the activation of specific genes in a variety of cell types under disparate physiological circumstances (Decker and Kovarik, 2000). Unphosphorylated STAT-1 is also known to play a crucial role in sustaining the constitutive induction or repression of numerous genes (Chatterjee-Kishore et al., 2000), and phosphorylation is dispensable for its nuclear localization in cells of different lineage (Chatterjee-Kishore et al., 2000; Meyer et al., 2003). A previous study on MUC4 promoter (Perrais et al., 2001) and additional in silico analyses of MUC4 promoter sequence by us (unpublished data) have identified putative binding sites for several transcription factors, including STAT, indicating IFN-γ as a potent regulator of MUC4 expression.

In the present study, we have investigated IFN-γ-induced expression of MUC4 in the pancreatic adenocarcinoma cell line, CD18/HPAF. We demonstrate that the sequential activation and upregulation of STAT-1 by IFN-γ drive the selective induction of IRF-1 (early response) and MUC4 (delayed response) within a single cell system. We further report that the stimulation of STAT-1 expression by double-stranded (ds)-polynucleotides also leads to MUC4 induction. Our findings expose a novel mechanism for IFN-γ response and unfold a new perspective on the regulation of STAT-1-induced gene expression.

Results

Induction of the MUC4 mucin gene by IFN-γ

To study the regulation of MUC4, we have used a two-component pancreatic tumour cell model comprised of the parental cell line, CD18/HPAF that produces relatively high levels of MUC4 and its serum-free-adapted derivative, CD18/HPAF-SF, which expresses low to undetectable endogenous MUC4 (Choudhury et al., 2000). On the basis of previously published data on MUC4 promoter structure (Perrais et al., 2001), we screened systematically a panel of potential regulators of this gene (unpublished data) and identified IFN-γ as a potent MUC4 inducer in CD18/HPAF-SF cells (Figure 1). RNA slot blot and western blot analyses revealed that MUC4 induction by IFN-γ was dose- and time-dependent in CD18/HPAF-SF cells, at both the mRNA (Figure 1a and c, respectively) and protein levels (Figure 1b and d). The earliest time point at which MUC4 upregulation could be detected was between 1 and 3 h post-treatment (Figure 1c and d, respectively).

Figure 1
figure1

Dose–response assay and time-course of MUC4 induction by IFN-γ. (a) Total RNA (10 μg/sample in triplicate) was analysed by RNA slot blot using 32P-labelled cDNA probes specific for MUC4 or GAPDH (internal control). The bar graph represents the intensity of MUC4-specific signal adjusted to GAPDH. Error bars indicate the standard deviation from triplicate values. (b) Total protein was processed for SDS–agarose (MUC4) or SDS–PAGE (phosphoglycerate kinase (PGK)) western blot analysis using antibodies specific for MUC4 or PGK. CD18/HPAF cells were used as a positive control for MUC4 expression. For the time-course of MUC4 induction, CD18/HPAF-SF cells were treated with IFN-γ (50 ng/ml) and harvested at the indicated time points after treatment. The bar graph represents the relative intensity of MUC4-specific signal adjusted to PGK. Error bars indicate the standard error from triplicate values. (c) Total RNA was analysed by RNA slot blotting as in (a). (d) Total protein was processed for SDS–agarose western blot analysis as in (b). The bar graph represents the relative intensity of MUC4-specific signal adjusted to PGK. Error bars indicate the standard error from triplicate values.

Role of the JAK/STAT pathway in MUC4 induction

The canonical Janus kinase (JAK)/STAT-1 pathway is the most common signalling route through which IFN-γ potentiates its pleiotropic activity (Boehm et al., 1997). Activation of this pathway follows the sequential phosphorylation of the receptor-associated tyrosine kinases JAK-1 and JAK-2, the IFN-γ receptor subunit 1 (IFNGR-1) and ultimately STAT-1, leading to its nuclear translocation and binding to responsive gene elements (Shuai et al., 1993). The two subunits of the multimeric IFN-γ receptor (IFNGR-1 and IFNGR-2) were constitutively expressed in CD18/HPAF-SF cells (Figure 2a). Moreover, western blot analyses showed a rapid Y701 phosphorylation of STAT-1 at 5 min post-treatment with IFN-γ (Figure 2b, top panel), reflecting the involvement of functional JAKs. Following activation, phospho-Y701-STAT-1 (hereon pY701-STAT-1) level peaked transiently at 30 min, remained unchanged for up to 12 h post-stimulation and declined thereafter. In contrast to pY701-STAT-1, the amount of total STAT-1 began to rise steadily around 1–3 h post-stimulation, with a more pronounced increase beginning 12 h post-stimulation (Figure 2b). Importantly, this expression pattern was strikingly similar to that of MUC4 (Figure 1c).

Figure 2
figure2

Profiles of expression/activation of IFN-γ receptors and STAT-1 in CD18/HPAF-SF cells. (a) Expression of IFN-γ receptor (IFNGR) subunits. Total RNA was isolated from CD18/HPAF-SF cells and analysed by reverse transcription–PCR using primers specific for IFNGR-1, IFNGR-2 or RPL13A (internal control). (b) To investigate the profiles of STAT-1 activation and expression, CD18/HPAF-SF cells were treated with IFN-γ (50 ng/ml) and harvested at the indicated time points after treatment. Total protein (30 μg per sample) was subjected to western blotting using anti-STAT-1 and anti-pY701-STAT-1 (activated STAT-1) antibodies. The bar graph represents the relative intensity of STAT-1- and pY701-STAT-1-specific signal adjusted to PGK. Error bars indicate the standard error from triplicate values.

Following IFN-γ stimulation, IRF-1 expression increased in a dose- and time-dependent manner (Figure 3a and b, respectively) and the time course paralleled with that of pY701-STAT-1 (Figure 2b, top row), while it differed from MUC4 both temporally and quantitatively. IRF-1 was upregulated rapidly within 15–30 min (Figure 3b), as is typically observed with immediate-early response genes, and its transcript level peaked at 6–12 h and declined thereafter (Figure 3b). By contrast, MUC4 was upregulated slowly between 1 and 3 h and continued to increase further up to 48 h post-stimulation (Figure 1c and d). Altogether, these observations indicate that the JAK/STAT pathway is operational in CD18/HPAF-SF cells; however, the disparity in the overall profiles of IRF-1 and MUC4 expression suggests the implication of distinct mechanisms in the regulation of these genes.

Figure 3
figure3

Dose–response and time-course of IRF-1 induction by IFN-γ. (a) CD18/HPAF-SF cells were exposed to the indicated doses of IFN-γ for 48 h. Total RNA (10 μg/sample in triplicate) was analysed by RNA slot blotting. (b) CD18/HPAF-SF cells were treated with IFN-γ (50 ng/ml) and harvested at the indicated time points after treatment. Total RNA was analysed by RNA slot blotting. The bar graph represents the relative intensity of IRF-1-specific signal adjusted to GAPDH. Error bars indicate the standard deviation from triplicate values.

Requirement for STAT-1 in JAK-mediated MUC4 induction

To resolve the discrepancy associated with the kinetics of MUC4 and IRF-1 induction (Figures 1c and 3b, respectively), we evaluated the respective roles of JAKs and STAT-1. Exposure of cells to the JAK-specific inhibitor AG-490 (Meydan et al., 1996) resulted in a dose-dependent inhibition of IFN-γ-triggered MUC4 induction (Figure 4a, lanes 12–14, top panel) as compared to the controls (lanes 9–11). Notably, the inhibition of MUC4 induction correlated with a decreased STAT-1 expression (Figure 4a, lanes 12–14, middle panel). Likewise, treatment of cells with the broad-spectrum kinase inhibitor staurosporine yielded a similar effect (data not shown). Of interest, treatment of unstimulated cells with AG-490 also led to the downregulation of basal STAT-1 levels (Figure 4a, lanes 5–7, middle panel). Together, these findings indicate that activation of the JAK kinase(s) is implicated, at least in part, in MUC4 gene induction by IFN-γ, and is also required for both the constitutive and stimulated expression of STAT-1. To confirm further that STAT-1 is an essential mediator in IFN-γ-dependent MUC4 gene induction, CD18/HPAF-SF cells were transiently transfected with STAT-1-specific short-interfering RNAs (siRNAs). IFN-γ was applied to cells when STAT-1 was minimally expressed, that is, 72 h post-transfection (Figure 4b, lane 4). Inhibition of STAT-1 by RNA-mediated interference (RNAi) decreased significantly IFN-γ-induced MUC4 expression (Figure 4b, lane 12) compared to the control (that is, TransIT-TKO+IFN-γ (lane 10)). In contrast, treatment of cells with luciferase-specific siRNA did not alter MUC4 expression either in the absence (lane 7) or the presence (lane 11) of IFN-γ. Thus, these data indicate that the inhibitory effect is specific for STAT-1 and support a critical role of this transcription factor in IFN-γ-dependent MUC4 induction.

Figure 4
figure4

Dissection of the JAK/STAT signalling pathway in CD18/HPAF-SF cells. (a) JAK inhibition. CD18/HPAF-SF cells were stimulated with IFN-γ (50 ng/ml) for 12 h in the presence of the indicated doses of AG-490 or equivalent volumes of dimethylsulphoxide (solvent). Total protein (50 μg per sample for MUC4; 30 μg per sample for STAT-1 and PGK) was analysed by western blotting. (b) STAT-1 knockdown by RNAi. CD18/HPAF-SF cells were transfected transiently with STAT-1-specific siRNA and exposed to IFN-γ (50 ng/ml) at 72 h post-transfection. Cells were harvested at the time of IFN-γ application or at 12 h post-treatment and analysed by western blotting as in (a). TKO, TransIT-TKO; Lucif., luciferase-specific siRNA. Total protein (30 μg per sample) was assayed by western blotting using anti-MUC4, anti-STAT-1 and anti-PGK antibodies.

MUC4 induction via a non-canonical mechanism

To identify the role of STAT-1 S727 phosphorylation in MUC4 induction, we examined the level of phospho-S727-STAT-1 (pS727-STAT-1) in resting and IFN-γ-treated cells. Stimulation by IFN-γ led to an upregulation of pS727-STAT-1-specific signals (Figure 5a) in parallel with an enhanced expression of total STAT-1. Upon stimulation with IFN-γ, the dynamics of STAT-1 subcellular distribution revealed three distinct phases, as they relate to gene activation. The first phase featured a nuclear accumulation of Y701- and S727-phosphorylated STAT-1 by 15 min post-treatment (Figure 5b, lane 2). This was associated with STAT-1 binding to the promoter region of IRF-1 (Figure 6b; GASIRF-1) as well as the distal region of MUC4 promoter (Perrais et al., 2001) (Figure 6b; GAS-IVMUC4 and GAS-VMUC4). Surprisingly, while STAT-1 binding resulted in the induction of the IRF-1 gene (15–30 min post-treatment) (Figure 3b), it did not affect MUC4 expression (Figure 1c). This was followed by a second phase that highlighted the increase in STAT-1 and MUC4 levels at 1.5 h (Figure 5b, lane 5), while the levels of both Y701- and S727-phosphorylated STAT-1 remained steady. During this phase, the binding of STAT-1 to the proximal region of MUC4 promoter (Figure 6b; GAS-IMUC4 and GAS-IIMUC4), and the induction of MUC4 (beginning 1.5–3.0 h post-stimulation) were observed. In the third phase, a gradual decline in the level of pY701-STAT-1 between 3.0 and 48 h was recorded (Figure 5, lanes 6–9), while the level of pS727-STAT-1 was sustained. During this phase, the level of total nuclear STAT-1 and cytoplasmic MUC4 continued to accumulate. Together, these data suggest that upregulation of STAT-1 plays a critical role in MUC4 gene induction; however, it does not exclude the possibility that the serine or tyrosine phosphorylation may, in part, also be implicated. This is in contrast to the canonical regulation of IRF-1, which relies primarily upon STAT-1 phosphorylation (Boehm et al., 1997). It should be noted, however, that the time course of STAT-1 interaction with the MUC4 promoter displayed a directional and progressive pattern, with the early and late interactions occurring in the distal (Figure 6b; GAS-IVMUC4 and GAS-VMUC4) and proximal (GAS-IMUC4 and GAS-IIMUC4) regions, respectively. This observation points to an alternative mechanism whereby the sequential unwinding of chromatin from the distal towards the proximal region of the MUC4 promoter is necessary for the activation of this gene.

Figure 5
figure5

Dynamics of STAT-1 subcellular distribution. (a) Analysis of STAT-1 serine 727 (S727) phosphorylation. CD18/HPAF-SF cells were treated with IFN-γ (50 ng/ml) and harvested at indicated time intervals. Total protein (30 μg per sample) was assayed by western blotting using pS727-STAT-1 and STAT-1 antibodies. The bar graph represents the relative intensity of STAT-1- and pS727-specific signal adjusted to PGK. Error bars indicate the standard error from triplicate values. (b) Biochemical assays. CD18/HPAF-SF cells were treated with IFN-γ (50 ng/ml), harvested at the indicated times post-treatment and subjected to subcellular fractionation. Cytoplasmic (30 μg per sample) and nuclear (150 μg per sample) extracts were analysed by western blotting. The bar graph represents the relative intensity of STAT-1-, pY701-STAT-1- and pS727-STAT-1-specific signal adjusted to PARP (in nuclear extracts) or relative intensity of MUC4-specific signal adjusted to PGK (top panel, in cytoplasmic extracts). Error bars indicate the standard error from triplicate values.

Figure 6
figure6

Profiles of STAT-1 interactions with the regulatory sequences of its target genes. (a) Structures of the promoter regions of the MUC4 and IRF-1 genes. Shown are the locations of putative GAS elements relative to the respective ATG translation start sites. Arrows indicate the transcription start sites. Diagrams are drawn to scale. (b) ChIP assay. CD18/HPAF-SF cells were treated with IFN-γ (50 ng/ml) and harvested at the indicated time points after treatment. Cells were processed for ChIP analysis using anti-STAT1 rabbit pAb and MUC4- and IRF-1-specfic primers flanking different GAS sites as described in Materials and methods. Rabbit IgG was used as a negative control in pull-down assays.

Relevance of STAT-1 upregulation to MUC4 induction

To determine further whether the IFN-γ-mediated upregulation of MUC4 depends primarily on the enhanced expression of STAT-1, we attempted to overexpress wild-type STAT-1 in CD18/HPAF cells by means of transient transfection. However, consistent with previous findings (Suzuki et al., 1999), the STAT-1 construct as well as an unrelated plasmid DNA (pGL3) induced the endogenous expression of STAT-1 (Figure 7a, lanes 4 and 6). The ds-polynucleotides (ds-DNA or ds-RNA)-induced upregulation of genes, including STAT-1, has been proposed to serve as the basis for an immunologically oriented response of non-immune cells to viral infection. Of note, the induction of STAT-1 by ds-polynucleotides is cell type- and nucleotide sequence-independent (Suzuki et al., 1999). As shown in Figure 7a, the increase in STAT-1 expression correlated with MUC4 induction (lanes 4 and 6). Although no tyrosine phosphorylation of the induced STAT-1 was observed, it was serine-phosphorylated (Figure 7a). To confirm further the direct implication of STAT-1 upregulation in MUC4 induction, cells were treated with STAT-1-specific siRNA, followed by transfection with plasmid DNA in such a way that STAT-1 upregulation by exogenous DNA fell within the range of its maximal inhibition by RNAi. In line with our expectation, MUC4 upregulation by plasmid DNA (Figure 7b, lane 11) was proportionately negated by siRNA-driven STAT-1 inhibition (lane 12). A residual induction of MUC4, however, was observed in the siRNA-treated sample, which may be associated with either a stimulation of the non-transfected cell population, a leaky inhibition by RNAi due to a strong induction by ds-polynucleotides in dually transfected cells, or a combination of both. Together, these data provide evidence for a role of STAT-1 upregulation in MUC4 induction.

Figure 7
figure7

Role of STAT-1 in ds-polynucleotide-induced MUC4 upregulation. (a) STAT-1 and MUC4 induction by ds-polynucleotides. CD18/HPAF-SF cells were transiently transfected with pGL3 Basic or 816W1 (STAT-1) plasmid constructs and harvested at 48 h post-transfection. Total protein (100 μg per sample (for MUC4); 30 μg per sample (for STAT-1, pY701-STAT-1, and PGK)) was analysed by western blotting. Lane 7, extract from cells treated with IFN- γ for 30 min used as a positive control for pY701-STAT-1. (b) STAT-1 knock-down by RNAi. CD18/HPAF-SF cells were transiently transfected with STAT-1-specific siRNA, re-transfected with 816W1 plasmid 54 h after siRNA transfection and harvested 30 h thereafter. Total protein was analysed by western blotting as in (a). Extract from cells treated with IFN-γ for 30 min was used as a positive control for pY701-STAT-1 (lane 13).

Discussion

IFN-γ is a pluripotent cytokine with a wide array of biological activities ranging from the modulation of immune responses to the regulation of cell proliferation and apoptosis (Boehm et al., 1997). While providing important information about mechanisms that regulate the IFN-γ-induced expression of the tumour-associated mucin gene, MUC4, our study has led us to the discovery of a hitherto unsuspected functional aspect of the ubiquitous transcription factor STAT-1. STAT-1 is known to play a key role in transducing signals mediated by a large number of cytokine and growth factor receptors (Darnell et al., 1994; Darnell, 1997). In previous studies, STAT-1-mediated induction of another membrane mucin, MUC1, has been reported in response to induction by IFN-γ (Lagow and Carson 2002). However, multiple observations in our study support the notion that upregulation of STAT-1 rather than its phosphorylation plays an important role in MUC4 induction by IFN-γ (Figures 1c and d, 2b, 4a and b, 5b, 6b and 7a and b), although the contribution of pY701- and/or pS727-STAT-1 to the induction process cannot be fully excluded (Figures 5b, 6b and 7a). In a recent study, STAT-3 upregulation was shown to be responsible for the transactivation of various genes in a tyrosine phosphorylation-independent manner (Yang et al., 2005). Moreover, enhanced expression of STAT-1and STAT-2 has been observed in IFN-γ-treated pancreatic carcinoid cells and was proposed to explain the anti-tumour effect of IFN-α (Zhou et al., 2001). Thus, it is likely that the ability to regulate gene expression via this mechanism may be a property shared by STAT family members. While the exact physiological implications of STATs’ upregulation are yet to be investigated, it can be predicted that it may impart enhanced diversity, specificity and efficacy to their gene regulatory properties. Collectively, these findings bring a significant improvement to our current knowledge about the molecular mechanisms controlling STAT-dependent gene expression.

The exact molecular events associated with gene transactivation via upregulation of STAT-1 remain unclear. Nonetheless, several lines of evidence support our findings and may account for the concurrent operation of the two STAT-1-dependent signalling pathways described herein. Previous studies have shown that the nuclear transport of pY701-STAT-1 and unphosphorylated STAT-1 occurs via independent routes, and that Y701 phosphorylation is dispensable for STAT-1 nuclear localization (Chatterjee-Kishore et al., 2000; Meyer et al., 2003). Moreover, other studies have demonstrated that unphosphorylated STAT-1 is capable of inducing or repressing the expression of various genes in a constitutive manner (Chatterjee-Kishore et al., 2000). Subsequent in vitro experiments designed to investigate the regulation of the low molecular mass polypeptide 2 (LMP2) gene revealed that, like STAT-1 in extracts of IFN-γ-stimulated cells, monomers and dimers of unphosphorylated recombinant STAT-1 could bind to DNA sequences harbouring half- and full-GAS sites, respectively (Chatterjee-Kishore et al., 2000). Furthermore, various reports have documented the ability of STAT-1 and STAT-3 to form homodimers before tyrosine phosphorylation (Mao et al., 2005). Of note, recent crystallographic data suggest that unphosphorylated STAT-1 dimers adopt a ‘parallel’ conformation, as is observed with DNA-bound pY701-STAT-1 dimers (Mao et al., 2005). In the light of these observations, we propose that upon upregulation, non-tyrosine-phosphorylated STAT-1 molecules accumulate in the nucleus (Figure 5b), whereby STAT-1 dimers interact with cognate GAS elements within the promoter regions of select target genes (for example, MUC4). The molecular basis of the differential responsiveness of target genes to either form of STAT-1 and/or the intrinsic selectivity associated with each isoform, however, remains elusive and warrants further investigations.

The genetic programmes associated with cellular responses to IFN-γ and other STAT-1-dependent cytokines involve a well-defined sequence of events. The initial part of the response features activation of immediate-early response genes whose induction is attributed to activated/phosphorylated STAT-1 molecules. This is followed by the activation of delayed early-/late-response genes that harbour regulatory cis-elements specific for the immediate-early response gene products. This cascade of events has been established for various cell systems (Boehm et al., 1997). Thus, our findings add a new aspect to this paradigm. The role of ligand-activated STAT-1-dependent pathway(s), which has been restricted mostly to the induction of immediate-early response genes, now extends to the regulation of a delayed early-/late-response gene, MUC4. On the basis of the data presented in this report, a model is proposed to recapitulate the current understanding of STAT-1-dependent gene regulation (Figure 8). This simplified model highlights the unique attributes of the newly identified signalling route and establishes a contrast between two pathways involving STAT-1 as a common gene transactivator. Of potential relevance to this model is the paradigm of ‘statosome’, according to which the bulk of cytoplasmic STAT molecules exist as part of large heteromeric complexes termed statosomes I and II (Guo et al., 2002). These complexes are involved in intracellular trafficking of STATs during cytokine signalling and are thought to sequester STAT molecules to the cytoplasmic compartment. Thus, we hypothesize that the upregulation of STAT-1 above a certain threshold may stoichiometrically titrate out statosome components, thereby enlarging the pool of ‘free/unbound’ cytosolic STAT-1 and facilitating its nuclear translocation. Alternatively, the dynamic, energy-independent, and carrier-free nuclear import of unphosphorylated STAT-1 (Marg et al., 2004), combined with its augmented availability in the cytoplasm (Figure 5b), may favour nuclear accumulation and, thus, the ability to regulate gene expression. A further clarification of these regulatory processes is warranted in the near future.

Figure 8
figure8

A model for STAT-1-dependent mechanisms of gene regulation. Highlighted in this model are the canonical (gray arrows) and non-canonical (red arrows) STAT-1-dependent signalling pathways that drive the selective induction of different target genes within a single cell system. Induction of the IRF-1 gene by IFN-γ implicates the canonical JAK/STAT pathway (Boehm et al., 1997) and relies primarily on Y701 phosphorylation of STAT-1. S727 phosphorylation of STAT-1 may involve the p38 mitogen-activated protein kinase (MAPK) (Goh et al., 1999). Regulation of the MUC4 gene depicts the novel STAT-1-dependent pathway, which is controlled by the level of STAT-1 protein and/or its phosphorylation at S727.

Materials and methods

Cell lines and cell culture reagents

CD18/HPAF-SF cells were maintained as discussed previously (Choudhury et al., 2000). All reagents for cell culture were purchased from Invitrogen (Carlsbad, CA, USA). Interferon-γ was obtained from Peprotech (Rocky Hill, NJ, USA).

Reverse transcription–PCR and slot blot analysis

Total RNA was isolated from cell lines using the RNeasykit (Qiagen Inc., Valencia, CA, USA). Reverse transcription (RT)–PCR and slot blot analyses were performed as described previously (Andrianifahanana et al., 2001).

Western blot analyses

Western blot analysis was performed as described previously (Singh et al., 2004). The primary antibodies used were anti-STAT-1áp91 (C-111) mAb (Santa-Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-pY701-STAT-1 pAb (Cell Signalling, Beverly, MA, USA), anti-pS727-STAT-1 pAb (Upstate, Lake Placid, NY, USA), and anti-PARP pAb Rabbit polyclonal antibody specific for phosphoglycerate kinase (PGK) was kindly provided by Dr Vishwanatha and used as an internal control.

Preparation and analysis of cytoplasmic and nuclear extracts

The protocol for cytoplasmic and nuclear extract preparation was adapted from previously published procedures (Lee and Green, 1990) with minor modifications of the hypotonic cytoplasmic extraction buffer and the hypertonic nuclear extraction buffer.

Chromatin immunoprecipitation analysis

Chromatin immunoprecipitation (ChIP) analysis was carried out as described previously (Nelson et al., 2006) with some modifications. Briefly, the cells were fixed with 0.8% formaldehyde for 10 min on ice followed by the addition of glycine to a final concentration of 125 mM. The cells were then washed with phosphate-buffered saline, harvested and subjected to sonication with 425–600 μm glass beads (Sigma, St Louis, MO, USA) in IP buffer (Nelson et al., 2006) utilizing an ultrasonic generator (Digital sonifier, Ultra Sonics Corp., Danburg, CT, USA). After centrifugation, the supernatant was diluted 10-fold with IP buffer and pre-cleared with Protein A-Sepharose (Pharmacia Biochemicals, Piscataway, NJ, USA) containing 20 μg/ml sheared salmon sperm DNA and 1 mg/ml bovine serum albumin. The pre-cleared chromatin fraction (50 μg of DNA) was incubated with anti-STAT1 pAb (BD Laboratories, Bedford, MA, USA) or rabbit IgG (Vector Laboratories, Burlingame, CA, USA) for 16 h at 4°C. Immune complexes were mixed with 50 μl of Protein A-Sepharose and washed sequentially with wash buffer (0.1% sodium dodecyl sulphate (SDS), 1% Triton X-100, 2 mM ethylenediamine tetraacetic acid (EDTA), 20 mM Tris-HCl, pH 8.1) containing 150 mM NaCl, wash buffer-1 containing 500 mM NaCl, wash buffer-2 (0.25 M LiCl, 1% NP40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1), and twice with TE (10 mM Tris-HCl, pH 8.1, 1 mM EDTA). The complexes were eluted in 1.0% SDS and 0.1 M NaHCO3 and crosslinking was reversed by heating to 65°C for 4 h. The fragmented DNA was purified and the GAS region(s) of IRF-1 and MUC4 genes (Table 1) were amplified by PCR using various sets of primers (Table 2). Amplified DNA was analysed in 3% agarose gel by electrophoresis.

Table 1 Characteristics of GAS elements within the promoter regions of MUC4 and IRF-1 genes
Table 2 PCR primers for ChIP assay

Knock-down by RNA interference

CD18/HPAF-SF cells were transiently transfected with STAT-1-specific double-stranded siRNA oligonucleotides – sense: 5′-IndexTermUCAGACAGUACCUGGCACA-dTdT-3′; antisense: 5′-IndexTermUGUGCCAGGUACUGUCUGA-dTdT-3′ (Dharmacon Research Inc., Chicago, IL, USA) – as per the manufacturer's recommendations using the TransIT-TKO reagent (Mirus Bio Corporation, Madison, WI, USA). Oligonucleotides specific for the luciferase gene – sense: 5′-IndexTermCGUACGCGGAAUACUUCGAdT-dT-3′; anti-sense: 5′-IndexTermUCGAAGUAUUCCGCGUACGdT-dT-3′ (Dharmacon Research Inc.) were used as a negative control.

Plasmid constructs and transfection

Endotoxin-free plasmid DNAs (wild-type STAT-1 (816W1) and pGL3) were transfected transiently into CD18/HPAF-SF cells using the FuGENE 6 transfection reagent (Roche Diagnostic, Indianapolis, IN, USA). Transfections were carried out according to the manufacturer's instructions. Cells were harvested at the indicated times post-transfection and processed for western blotting as described above. For staggered transfection experiments (that is, siRNA followed by plasmid), cells were briefly trypsinized (4 min) without a loss of adherence before the second transfection.

Inhibition assays

For analysis of Janus kinase (JAK)-dependent signalling, cells were pretreated with the JAK-specific inhibitor AG-490 (A.G. Scientific Inc., San Diego, CA, USA) before stimulation with IFN-γ. AG-490 was applied to cells at the indicated concentrations for 30 min and replenished at the time of IFN-γ treatment. Cells were incubated in the presence of the inhibitor throughout the experiments. Cells were harvested at 12 h post-treatment and processed for total protein extraction and western blot analysis as described above.

Accession codes

Accessions

GenBank/EMBL/DDBJ

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Acknowledgements

This work was supported, in part, by an R01 grant CA78590 from the National Institutes of Health. We are grateful to Dr Robert Arceci (Johns Hopkins University) for providing the STAT-1 plasmid construct. We also thank Kristi LW Berger (Eppley Institute) for editorial assistance. This work was supported by an RO1 grant CA78590 from the National Institutes of Health.

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Correspondence to S K Batra.

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Andrianifahanana, M., Singh, A., Nemos, C. et al. IFN-γ-induced expression of MUC4 in pancreatic cancer cells is mediated by STAT-1 upregulation: a novel mechanism for IFN-γ response. Oncogene 26, 7251–7261 (2007). https://doi.org/10.1038/sj.onc.1210532

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Keywords

  • IFN-γ
  • MUC4
  • STAT-1
  • pancreatic cancer
  • regulation

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