EGFR-targeted mAb therapy modulates autophagy in head and neck squamous cell carcinoma through NLRX1-TUFM protein complex.

Epidermal growth factor receptor (EGFR)-targeted therapy in head and neck squamous cell carcinoma (HNSCC) patients frequently results in tumor resistance to treatment. Autophagy is an emerging underlying resistance mechanism, however, the molecular autophagy machinery in HNSCC cells and potential biomarkers of patient response to EGFR-targeted therapy remain insufficiently characterized. Here we show that the EGFR blocking with cetuximab leads to varied autophagic responses, which modulate cancer cell susceptibility to EGFR inhibition. Inhibition of autophagy sensitizes HNSCC cells to EGFR blockade. Importantly, we identify a novel signaling hub centering on the NLRX1 (nucleotide-binding, lots of leucine-rich repeats-containing protein member X1)-TUFM (Tu translation elongation factor mitochondrial) protein complex, promoting autophagic flux. Defects in the expression of either NLRX1 or TUFM result in compromised autophagy when treated with EGFR inhibitors. As a previously undefined autophagy-promoting mechanism, we found that TUFM serves as a novel anchorage site, recruiting Beclin-1 to mitochondria, promoting its polyubiquitination, and interfering with its interaction with Rubicon. This protein complex is also essential for endoplasmic reticulum stress signaling induction, possibly as an additional mechanism to promote autophagy. Utilizing tumor specimens from a novel neoadjuvant clinical trial, we show that increased expression of the autophagy adaptor protein, SQSTM1/p62, is associated with poor response to cetuximab therapy. These findings expand our understanding of the components involved in HNSCC autophagy machinery that responds to EGFR inhibitors, and suggest potential combinatorial approaches to enhance its therapeutic efficacy.

ASC deficiency suppresses proliferation and prevents medulloblastoma incidence.

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is silenced by promoter methylation in many types of tumors, yet ASC's role in most cancers remains unknown. Here, we show that ASC is highly expressed in a model of medulloblastoma, the most common malignant pediatric brain cancer; ASC is also expressed in human medulloblastomas. Importantly, while ASC deficiency did not affect normal cerebellar development, ASC knockout mice on the Smoothened (ND2:SmoA1) transgenic model of medulloblastoma exhibited a profound reduction in medulloblastoma incidence and a delayed tumor onset. A similar decrease in tumorigenesis with ASC deficiency was also seen in the hGFAP-Cre:SmoM2 mouse model of medulloblastoma. Interestingly, hyperproliferation of the external granule layer (EGL) was comparable at P20 in both wild-type and ASC-deficient SmoA1 mice. However, while the apoptosis and differentiation markers remained unchanged at this age, proliferation makers were decreased, and the EGL was reduced in thickness and area by P60. This reduction in proliferation with ASC deficiency was also seen in isolated SmoA1 cerebellar granule precursor cells in vitro, indicating that the effect of ASC deletion on proliferation was cell autonomous. Interestingly, ASC-deficient SmoA1 cerebella exhibited disrupted expression of genes in the transforming growth factor-ß pathway and increased level of nuclear Smad3. Taken together, these results demonstrate an unexpected role for ASC in Sonic hedgehog-driven medulloblastoma tumorigenesis, thus identifying ASC as a promising novel target for antitumor therapy.

ASC-dependent RIP2 kinase regulates reduced PGE2 production in chronic periodontitis.

Levels of prostaglandin E(2) (PGE(2)) and its processing enzyme, prostaglandin-endoperoxide-synthase-2/ cyclooxygenase-2 (PTGS2/COX-2), are elevated in actively progressing periodontal lesions, but suppressed in chronic disease. COX-2 expression is regulated through inflammatory signaling that converges on the mitogen-activated protein kinase (MAPK) pathway. Emerging evidence suggests a role for the inflammatory adaptor protein, ASC/Pycard, in MAPK activation. We postulated that ASC may represent a mediator of the MAPK-mediated regulatory network of PGE(2) production. Using RNAi-mediated gene slicing, we demonstrated that ASC regulates COX-2 expression and PGE(2) production in THP1 monocytic cells following infection with Porphyromonas gingivalis (Pg). Production of PGE(2) did not require the inflammasome adaptor function of ASC, but was dependent on MAPK activation. Furthermore, the MAP kinase kinase kinase CARD domain-containing protein RIPK2 was induced by Pg in an ASC-dependent manner. Reduced ASC and RIPK2 levels were revealed by orthogonal comparison of the expression of the RIPK family in ASC-deficient THP1 cells with that in chronic periodontitis patients. We show that pharmacological inhibition of RIPK2 represses PGE(2) secretion, and RNAi-mediated silencing of RIPK2 leads to diminished MAPK activation and PGE(2) secretion. These findings identify a novel ASC-RIPK2 axis in the generation of PGE(2) that is repressed in patients diagnosed with chronic adult periodontitis.

Combined donor specific transfusion and anti-CD154 therapy achieves airway allograft tolerance.

BACKGROUND: The state of tolerance allows long term graft survival without immunosuppressants. Lung transplantation tolerance has not been consistently achieved in either small or large animal models. METHODS: The mechanisms and effectiveness of a tolerance induction protocol consisting of donor specific transfusion (DST; day 0) and a short course of co-stimulatory blockade (anti-CD154 antibody; days -7, -4, 0 and +4) were studied in the mouse heterotopic tracheal transplant model of chronic lung rejection. C57BL/6 mice received BALB/c tracheal grafts (day 0) and were treated with DST alone, anti-CD154 alone, the combination (DST/anti-CD154), or no treatment. No non-specific immunosuppressants were used. RESULTS: DST/anti-CD154 in combination, but neither treatment alone, markedly prolonged the lumen patency and survival (>100 days) of fully histo-incompatible allografts (p<0.05 versus control allografts at every time point studied up to 16 weeks) without immunosuppression. This protocol was donor antigen specific as third party grafts (C3H) were promptly rejected. In addition, DST/anti-CD154 did not result in mixed chimerism but induced transplantation tolerance via a peripheral mechanism(s), which included significantly reduced cytotoxic T cell activity (p<0.001) and a significantly increased percentage of CD4+CD25+ cells (p = 0.03). CONCLUSIONS: The DST/anti-CD154 protocol successfully induced and maintained long term, donor specific tolerance in the mouse heterotopic airway graft model of chronic lung rejection. This finding may lead us closer to successful tolerance induction in lung transplantation.

Identification of novel genes and transcription factors involved in spleen, thymus and immunological development and function.

We constructed and analyzed six serial analysis of gene expression (SAGE) libraries to identify genes with previously uncharacterized roles in spleen or thymus development. A total of 625 070 tags were sequenced from the three spleen (embryonic day (E)15.5, E16.5 and adult) and three thymus (E15.5, E18.5 and adult) libraries. These tags corresponded to 83 182 tag types, which mapped unambiguously to 36 133 different genes. Genes over-represented in these libraries, compared to 115 mouse SAGE libraries (www.mouseatlas.org), included genes of known and unknown immunological or developmental relevance. The expression profiles of 11 genes with unknown roles in spleen and thymus development were validated using reverse transcription-qPCR. We further characterized the expression of one of these candidates, RIKEN cDNA 9230105E10 that encodes a murine homolog of Trim5alpha, in numerous adult tissues and immune cell types. In addition, we demonstrate that transcript levels are upregulated in response to TLR stimulation of plasmacytoid dendritic cells and macrophages. This work provides the first evidence of regulated and cell type-specific expression of this gene. In addition, these observations suggest that the SAGE libraries provide an important resource for further investigations into the molecular mechanisms regulating spleen and thymus organogenesis, as well as the development of immunological competence.

A comparison of signaling activities induced by Taxol and desoxyepothilone B.

Desoxyepothilone B (dEpoB), currently in clinical trials, is a novel microtubule inhibitor with similar mode-of-action to paclitaxel (Taxol). Intriguingly, it is effective in some cell lines and tumor xenografts refractory to Taxol. The purpose of this study is to compare signaling induced by the two drugs and identify a molecular basis for increased efficacy of dEpoB in resistant lines. The importance of ERK signaling, already established for Taxol, was shown for dEpoB and other G2-blocking agents. However, a role in differential sensitivity was not observed. Affymetrix analysis shows similar gene modulation by either agent, alone or in combination with MEK inhibitor. Differential sensitivity in a set of Taxol-resistant lines correlated to the expression of P-glycoprotein (P-gp), and its importance was demonstrated directly. These results suggest that Taxol and dEpoB elicit similar cell death pathways, and the increased efficacy of dEpoB in resistant tumor lines lies in differential susceptibility to P-gp.

Inhibition of IFN-gamma signaling by an Epstein-Barr virus immediate-early protein.

Viruses have evolved elaborate mechanisms to target many aspects of the host's immune response. The cytokine IFN-gamma plays a central role in resistance of the host to infection via direct antiviral effects as well as modulation of the immune response. In this study, we demonstrate that the Epstein-Barr virus (EBV) immediate-early protein, BZLF1, inhibits the IFN-gamma signaling pathway. BZLF1 decreases the ability of IFN-gamma to activate a variety of important downstream target genes, such as IRF-1, p48, and CIITA, and prevents IFN-gamma-induced class II MHC surface expression. Additionally, BZLF1 inhibits IFN-gamma-induced STAT1 tyrosine phosphorylation and nuclear translocation. Finally, we demonstrate that BZLF1 decreases expression of the IFN-gamma receptor, suggesting a mechanism by which EBV may escape antiviral immune responses during primary infection.

TNF alpha promotes proliferation of oligodendrocyte progenitors and remyelination.

Here we used mice lacking tumor necrosis factor-alpha (TNF alpha) and its associated receptors to study a model of demyelination and remyelination in which these events could be carefully controlled using a toxin, cuprizone. Unexpectedly, the lack of TNF alpha led to a significant delay in remyelination as assessed by histology, immunohistochemistry for myelin proteins and electron microscopy coupled with morphometric analysis. Failure of repair correlated with a reduction in the pool of proliferating oligodendrocyte progenitors (bromodeoxyuridine-labeled NG2(+) cells) followed by a reduction in the number of mature oligodendrocytes. Analysis of mice lacking TNF receptor 1 (TNFR1) or TNFR2 indicated that TNFR2, not TNFR1, is critical to oligodendrocyte regeneration. This unexpected reparative role for TNF alpha in the CNS is important for understanding oligodendrocyte regeneration/proliferation, nerve remyelination and the design of new therapeutics for demyelinating diseases.

Mechanisms of nuclear import and export that control the subcellular localization of class II transactivator.

The presence of the class II transactivator (CIITA) activates the transcription of all MHC class II genes. Previously, we reported that deletion of a carboxyl-terminal nuclear localization signal (NLS) results in the cytoplasmic localization of CIITA and one form of the type II bare lymphocyte syndrome. However, further sequential carboxyl-terminal deletions of CIITA resulted in mutant forms of the protein that localized predominantly to the nucleus, suggesting the presence of one or more additional NLS in the remaining sequence. We identified a 10-aa motif at residues 405-414 of CIITA that contains strong residue similarity to the classical SV40 NLS. Deletion of this region results in cytoplasmic localization of CIITA and loss of transactivation activity, both of which can be rescued by replacement with the SV40 NLS. Fusion of this sequence to a heterologous protein results in its nuclear translocation, confirming the identification of a NLS. In addition to nuclear localization sequences, CIITA is also controlled by nuclear export. Leptomycin B, an inhibitor of export, blocked the nuclear to cytoplasmic translocation of CIITA; however, leptomycin did not alter the localization of the NLS mutant, indicating that this region mediates only the rate of import and does not affect CIITA export. Several candidate nuclear export sequences were also found in CIITA and one affected the export of a heterologous protein. In summary, we have demonstrated that CIITA localization is balanced between the cytoplasm and nucleus due to the presence of NLS and nuclear export signal sequences in the CIITA protein.

Downregulation of CIITA function by protein kinase a (PKA)-mediated phosphorylation: mechanism of prostaglandin E, cyclic AMP, and PKA inhibition of class II major histocompatibility complex expression in monocytic lines.

Prostaglandins, pleiotropic immune modulators that induce protein kinase A (PKA), inhibit gamma interferon induction of class II major histocompatibility complex (MHC) genes. We show that phosphorylation of CIITA by PKA accounts for this inhibition. Treatment with prostaglandin E or 8-bromo-cyclic AMP or transfection with PKA inhibits the activity of CIITA in both mouse and human monocytic cell lines. This inhibition is independent of other transcription factors for the class II MHC promoter. These same treatments also greatly reduced the induction of class II MHC mRNA by CIITA. PKA phosphorylation sites were identified using site-directed mutagenesis and phosphoamino acid analysis. Phosphorylation at CIITA serines 834 and 1050 accounts for the inhibitory effects of PKA on CIITA-driven class II MHC transcription. This is the first demonstration that the posttranslational modification of CIITA mediates inhibition of class II MHC transcription.

Constitutive expression of MHC class II genes in melanoma cell lines results from the transcription of class II transactivator abnormally initiated from its B cell-specific promoter.

In melanoma cell lines, two different patterns of MHC class II expression have been described, either an IFN gamma-inducible expression of HLA-DR and HLA-DP, with a faint or null expression of HLA-DQ, resembling that described for melanocytes, or a constitutive expression, i.e., IFN-gamma independent, of all three HLA-D isotypes. As this latter phenotype has been associated with a more rapid progression of melanoma tumors, we have analyzed in different melanoma cell lines the molecular mechanisms leading to this abnormal pattern of MHC class II expression. In agreement with the evidence of a coordinate transcription of the HLA-D genes in these cell lines, we have shown the constitutive expression of CIITA (class II transactivator) transcripts, CIITA being known as the master switch of MHC class II expression. Unexpectedly, these transcripts initiate from promoter III of the CIITA gene, a promoter that is mainly used constitutively in B lymphocytes. This expression was further shown to occur through factor(s) acting on the enhancer located upstream of CIITA promoter III, which was previously described in epithelioid cells as an IFN-gamma-response sequence. The hypothesis of a general abnormality of the IFN-gamma transduction pathway was dismissed. Constitutive transcription of CIITA from promoter III having been observed in unrelated melanoma cell lines, we propose the hypothesis that this phenomenon might not be a random event, but could be linked to the neoplasic state of the melanoma cells.

Identification of novel Mycoplasma hyorhinis gene fragments by differential display analysis of co-cultures.

Mycoplasmas are a diverse group of wall-less prokaryotes that have evolved an unusually small genome by adopting a parasitic mode of life. Recently, intense efforts have been made to sequence mycoplasma genomes and to define a minimal genome using mycoplasma as a model. Due to their parasitic nature, mycoplasma species are often difficult to cultivate, making it challenging to identify and sequence mycoplasma genes. In this report, we describe a method for identifying mycoplasma gene fragments from co-cultures using differential display analysis. Using this technique, we have identified fragments of seven putative genes from Mycoplasma hyorhinis. Sequence similarities suggest that four of these genes are members of the proposed minimal mycoplasma genome. The application of differential display analysis to co-cultures should be useful in the identification of genes from a variety of pathogenic organisms that are difficult to cultivate without a host.

MEK inhibition enhances paclitaxel-induced tumor apoptosis.

The anti-cancer drug paclitaxel (Taxol) alters microtubule assembly and activates pro-apoptotic signaling pathways. Previously, we and others found that paclitaxel activates endogenous JNK in tumor cells, and the activation of JNK contributes to tumor cell apoptosis. Here we find that paclitaxel activates the prosurvival MEK/ERK pathway, which conversely may compromise the efficacy of paclitaxel. Hence, a combination treatment of paclitaxel and MEK inhibitors was pursued to determine whether this treatment could lead to enhanced apoptosis. The inhibition of MEK/ERK with a pharmacologic inhibitor, U0126, together with paclitaxel resulted in a dramatic enhancement of apoptosis that is four times more than the additive value of the two drugs alone. Enhanced apoptosis was verified by the terminal transferase-mediated dUTP nick end labeling assay, by an enzyme-linked immunosorbent assay for histone-associated DNA fragments, and by flow cytometric analysis for DNA content. Specificity of the pharmacologic inhibitor was confirmed by the use of (a) a second MEK/ERK inhibitor and (b) a transdominant-negative MEK. Enhanced apoptosis was verified in breast, ovarian, and lung tumor cell lines, suggesting this effect is not cell type-specific. This is the first report of enhanced apoptosis detected in the presence of paclitaxel and MEK inhibition and suggests a new anticancer strategy.

How Stat1 mediates constitutive gene expression: a complex of unphosphorylated Stat1 and IRF1 supports transcription of the LMP2 gene.

Analysis of mRNA levels in cells that express or lack signal transducers and activators of transcription 1 (Stat1) reveals that Stat1 mediates the constitutive transcription of many genes. Expression of the low molecular mass polypeptide 2 (LMP2), which requires Stat1, has been studied in detail. The overlapping interferon consensus sequence 2/gamma-interferon-activated sequence (ICS-2/GAS) elements in the LMP2 promoter bind to interferon regulatory factor 1 (IRF1) and Stat1 and are occupied constitutively in vivo. The point mutant of Stat1, Y701F, which does not form dimers involving SH2-phosphotyrosine interactions, binds to the GAS element and supports LMP2 expression. Unphosphorylated Stat1 binds to IRF1 directly and we conclude that this complex uses the ICS-2/GAS element to mediate constitutive LMP2 transcription in vivo. The promoter of the IRF1 gene, which also contains a GAS site but not an adjacent ICS-2 site, is not activated by Stat1 Y701F. The promoters of other genes whose constitutive expression requires Stat1 may also utilize complexes of unphosphorylated Stat1 with IRF1 or other transcription factors.

Identification of class II transcriptional activator-induced genes by representational difference analysis: discoordinate regulation of the DN alpha/DO beta heterodimer.

Class II transcriptional activator (CIITA) is a master regulator of MHC class II genes, including DR, DP, and DQ, and MHC class II-associated genes DM and invariant chain. To determine the repertoire of genes that is regulated by CIITA and to identify uncharacterized CIITA-inducible genes, we used representational difference analysis. Representational difference analysis screens for differentially expressed transcripts. All CIITA-induced genes were MHC class II related. We have identified the alpha subunit, DN alpha, of the class II processing factor DO as an additional CIITA-inducible gene. Northern analysis confirmed that DN alpha is induced by IFN-gamma in 2fTGH fibrosarcoma cells, and CIITA is necessary for high-level expression in B cells. The beta subunit, DO beta, is not inducible in fibrosarcoma cells by IFN-gamma or exogenous CIITA expression. Moreover, in contrast to other class II genes, DO beta expression remains high in the absence of CIITA in B cells. The promoters for DN alpha and DO beta contain the highly conserved WXY motifs, and, like other class II genes, expression of both DN alpha and DO beta requires RFX. These findings demonstrate that both DN alpha and DO beta are regulated by RFX. However, DN alpha is defined for the first time as a CIITA-inducible gene, and DO beta as a MHC class II gene whose expression is independent of CIITA.

Paclitaxel up-regulates interleukin-8 synthesis in human lung carcinoma through an NF-kappaB- and AP-1-dependent mechanism.

Lung cancer is a leading cause of cancer-related death in the United States. For this reason we chose to study the specific cellular effects that one chemotherapeutic agent, paclitaxel, has on lung carcinoma. In addition to its known mechanism of action, which is to stabilize microtubules, paclitaxel has been shown to have other interesting and relevant cellular effects. In this report, we demonstrate that a subset of human lung carcinoma cell lines respond to paclitaxel treatment with an up to a fivefold increase in the production of interleukin-8 (IL-8). We demonstrate that this increased production is specific to IL-8 but not to other chemokines, and is both dose- and time-dependent. Increased IL-8 mRNA is seen as early as 45 min with a peak at 4 h after paclitaxel treatment. This increase in mRNA is due to transcriptional activation because actinomycin D treatment blocked the increase. Paclitaxel also activates the mitogen-activated protein kinase family member, JNK1, in dose-dependent fashion. IL-8 enhancement is completely abolished with the use of an inhibitor of NF-kappaB, the super-repressor IkappaB. Similar results were obtained upon the inhibition of AP-1 activation with the MEK1/2 inhibitor, U0126. By gaining a better understanding of the differences in cellular response to paclitaxel chemotherapy, these findings might lead to either improved patient selection or to the development of adjuvant therapy targeted at specific-cell signaling proteins.

IL-8 reduced tumorigenicity of human ovarian cancer in vivo due to neutrophil infiltration.

Paclitaxel is a frontline therapy for ovarian cancer. Our laboratory has shown that paclitaxel induces IL-8, a member of the C-X-C family of chemokines, in subsets of human ovarian cancer cells. However, the critical issue concerns the biological significance of this chemokine in human ovarian cancer. To study the influence of IL-8 on tumor growth, human ovarian cancer cell lines were transfected with an expression vector for human IL-8 and tested for their ability to form tumors in nude mice. IL-8 expression by the transfected cells did not alter their growth properties in vitro. In contrast, tumor growth in vivo was significantly attenuated in animals receiving IL-8-expressing cells when compared with mice injected with control cells. As additional evidence that IL-8 is a crucial factor in tumor growth, it was noted that ovarian cell lines in which constitutive IL-8 expression is elevated did not form tumors. Injection of neutralizing Ab to IL-8 reverted the phenotype and caused tumor growth in vivo. Examination of tissue from the inoculation site revealed a dramatically elevated cellularity, containing neutrophils and macrophages, in mice receiving IL-8-expressing tumor cells. These results suggest that IL-8 production by human ovarian tumor cells can play a role in reducing the rate of tumor growth; this effect may be mediated by the increased targeting of neutrophil and other mononuclear cells to the tumor injection site. These studies indicate a role for IL-8 in ovarian cancer control and suggest that chemotherapy-induced IL-8 may have a positive role in controlling tumor growth.

Analysis of the defect in IFN-gamma induction of MHC class II genes in G1B cells: identification of a novel and functionally critical leucine-rich motif (62-LYLYLQL-68) in the regulatory factor X 5 transcription factor.

MHC class II deficiency found in bare lymphocyte syndrome patients results from the absence or dysfunction of MHC class II transcriptional regulators, such as regulatory factor X (RFX) and class II transactivator (CIITA). Understanding the roles of these factors has been greatly facilitated by the study of genetic defects in cell lines of bare lymphocyte syndrome patients, as well as in cell lines that have been generated by chemical mutagenesis in vitro. The latter group includes MHC class II-deficient lines that are no longer responsive to induction by IFN-gamma. Here, we show that the defect in G1B, one such cell line, is attributed to the lack of functional RFX5, the largest subunit of RFX. The RFX5 gene isolated from G1B cells contains two separate single-base pair mutations. One alteration does not exhibit a phenotype, whereas a leucine-to-histidine mutation eliminates DNA-binding and transactivating functions. This mutation lies outside of previously defined functional domains of RFX5 but within an unusual, leucine-rich region (62-LYLYLQL-68). To further investigate the significance of the leucine-rich region, we targeted all neighboring leucine residues for mutagenesis. These mutants were also unable to transactivate a MHC class II reporter gene, confirming that these leucine residues play an essential role in RFX activity and characterize a novel leucine-rich motif.

A novel element and a TEF-2-like element activate the major histocompatibility complex class II transactivator in B-lymphocytes.

Major histocompatibility complex (MHC) class II molecules play a central role in immune responses, and transcription of this family of genes requires the MHC class II transactivator (CIITA). CIITA has four promoters, which are transcribed in a tissue-specific manner. CIITA promoter III is constitutively active in mature B-lymphocytes. This report now describes the minimal 319-base pair promoter region necessary for maximal transcriptional activity in B-lymphocytes. Ultraviolet light and dimethylsulfate in vivo genomic footprinting analyses reveal five occupied DNA sequence elements present in intact B-lymphocytes. Functional analysis of these elements using promoter deletions and site-specific mutations demonstrates that at least two of the sites occupied in vivo are critical for transcriptional activity. In vitro protein/DNA analysis suggests that one of the sites is a TEF-2-like element and the other is occupied by a novel transcription activator. In addition, nuclear factor-1 associates with the promoter both in vivo and in vitro. In myeloma cell lines, loss of CIITA transcription correlates with a completely unoccupied CIITA promoter III. These findings suggest that CIITA transcription in B-lymphocytes is activated through at least two strong promoter elements, while loss of expression in myeloma cells is mediated through changes in promoter assembly.

Differential selectivity of CIITA promoter activation by IFN-gamma and IRF-1 in astrocytes and macrophages: CIITA promoter activation is not affected by TNF-alpha.

During demyelinating disease of the central nervous system (CNS), locally elevated cytokine levels may induce upregulation of MHC class II molecules on otherwise low expressing or negative cell types such as microglia and astrocytes, since IFN-gamma has been shown to induce MHC class II expression on these cell types in vitro. While many transcription factors are involved with MHC class II expression, only the class II transactivator (CIITA) is tightly coordinated with IFN-gamma-inducibility. Control of CIITA gene expression is complex, involving four distinct promoters, two of which (promoters III and IV) are IFN-gamma-inducible in certain cell types. Here we demonstrate that IFN-gamma treatment of rat astrocytes induces only CIITA promoter IV activity in contrast to the murine macrophage cell line RAW 264.7 that uses both IFN-gamma-inducible promoters. In contrast to previously published reports, promoter IV activation is completely dependent upon an intact interferon regulatory factor-1 (IRF-1) but not STAT binding site using promoter constructs specifically mutated at these positions. Importantly, while TNF-alpha is able to synergize with IFN-gamma to increase astrocyte MHC class II expression in vitro, we show that treatment of rat astrocytes with TNF-alpha has no effect on CIITA promoter activity. These data demonstrate that TNF-alpha augments MHC class II expression through a mechanism downstream or independent of CIITA induction.