FANCC interacts with Hsp70 to protect hematopoietic cells from IFN-gamma/TNF-alpha-mediated cytotoxicity.

The Fanconi anemia (FA) complementation group C gene product (FANCC) functions to protect hematopoietic cells from cytotoxicity induced by interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and double-stranded RNA (dsRNA). Because apoptotic responses of mutant FA-C cells involve activation of interferon-inducible, dsRNA-dependent protein kinase PKR, we sought to identify FANCC-binding cofactors that may modulate PKR activation. We identified the molecular chaperone Hsp70 as an interacting partner of FANCC in lymphoblasts and HeLa cells using 'pull-down' and co-immunoprecipitation experiments. In vitro binding assays showed that the association of FANCC and Hsp70 involves the ATPase domain of Hsp70 and the central 320 residues of FANCC, and that both Hsp40 and ATP/ADP are required. In whole cells, Hsp70-FANCC binding and protection from IFN-gamma/TNF-alpha-induced cytotoxicity were blocked by alanine mutations located in a conserved motif within the Hsp70-interacting domain of FANCC. We therefore conclude that FANCC acts in concert with Hsp70 to prevent apoptosis in hematopoietic cells exposed to IFN-gamma and TNF-alpha.

The Fanconi anemia complementation group C gene product: structural evidence of multifunctionality.

The Fanconi anemia (FA) group C gene product (FANCC) functions to protect cells from cytotoxic and genotoxic effects of cross-linking agents. FANCC is also required for optimal activation of STAT1 in response to cytokine and growth factors and for suppressing cytokine-induced apoptosis by modulating the activity of double-stranded RNA-dependent protein kinase. Because not all FANCC mutations affect STAT1 activation, the hypothesis was considered that cross-linker resistance function of FANCC depends on structural elements that differ from those required for the cytokine signaling functions of FANCC. Structure-function studies were designed to test this notion. Six separate alanine-substituted mutations were generated in 3 highly conserved motifs of FANCC. All mutants complemented mitomycin C (MMC) hypersensitive phenotype of FA-C cells and corrected aberrant posttranslational activation of FANCD2 in FA-C mutant cells. However, 2 of the mutants, S249A and E251A, failed to correct defective STAT1 activation. FA-C lymphoblasts carrying these 2 mutants demonstrated a defect in recruitment of STAT1 to the interferon gamma (IFN-gamma) receptor and GST-fusion proteins bearing S249A and E251A mutations were less efficient binding partners for STAT1 in stimulated lymphoblasts. These same mutations failed to complement the characteristic hypersensitive apoptotic responses of FA-C cells to tumor necrosis factor-alpha (TNF-alpha) and IFN-gamma. Cells bearing a naturally occurring FANCC mutation (322delG) that preserves this conserved region showed normal STAT1 activation but remained hypersensitive to MMC. The conclusion is that a central highly conserved domain of FANCC is required for functional interaction with STAT1 and that structural elements required for STAT1-related functions differ from those required for genotoxic responses to cross-linking agents. Preservation of signaling capacity of cells bearing the del322G mutation may account for the reduced severity and later onset of bone marrow failure associated with this mutation.

Functional correction of FA-C cells with FANCC suppresses the expression of interferon gamma-inducible genes.

Because hematopoietic cells derived from Fanconi anemia (FA) patients of the C-complementation group (FA-C) are hypersensitive to the inhibitory effects of interferon gamma (IFNgamma), the products of certain IFNgamma-inducible genes known to influence hematopoietic cell survival were quantified. High constitutive expression of the IFNgamma-inducible genes, IFN-stimulated gene factor 3 gamma subunit (ISGF3gamma), IFN regulatory factor-1 (IRF-1), and the cyclin-dependent kinase inhibitor p21(WAF1) was found in FANCC mutant B lymphoblasts, low-density bone marrow cells, and murine embryonic fibroblasts. Paradoxically, these cells do not activate signal transducer and activator of transcription (STAT) 1 properly. In an attempt to clarify mechanisms by which FA-C cells overexpress IFNgamma-inducible genes in the face of defective STAT1 phosphorylation, it was reasoned that decreased levels of activated STAT1 might result in reduced expression of a hematopoietic IFNgamma-responsive protein that normally modulates expression of other IFNgamma-responsive genes. Levels of the IFNgamma-inducible factor IFN consensus sequence binding protein (ICSBP), a negative trans-acting regulator of some IFNgamma-inducible genes, were quantified. ICSBP levels were reduced in FA-C B lymphoblasts and MEFs. However, enforced expression of ICSBP failed to down-regulate IRF-1, ISGF3gamma, and p21(WAF1). Thus, the FANCC protein functions to modulate expression of a family of genes that in normal cells are inducible only by specific environmental cues for apoptosis or mitogenic inhibition, but it does so independently of the classic IFN-STAT1 pathway and is not the direct result of reduced ICSBP expression.

Role of double-stranded RNA-dependent protein kinase in mediating hypersensitivity of Fanconi anemia complementation group C cells to interferon gamma, tumor necrosis factor-alpha, and double-stranded RNA.

Hematopoietic cells bearing inactivating mutations of Fanconi anemia group C (FANCC) are excessively apoptotic and demonstrate hypersensitivity not only to cross-linking agents but also to interferon gamma (IFN-gamma) and tumor necrosis factor-alpha. Seeking essential signaling pathways for this phenotype, this study quantified constitutive and induced RNA-dependent protein kinase (PKR) activation in Fanconi anemia cells of the C complementation group (FA-C). PKR was constitutively phosphorylated and exhibited an increased binding affinity for double-stranded RNA (dsRNA) in FANCC(-/-) cells. FANCC(-/-) cells were hypersensitive to both dsRNA and the combination of dsRNA and IFN-gamma in that these agents induced a higher fraction of apoptosis in FANCC(-/-) cells than in normal cells. Overexpression of wild-type PKR-sensitized FANCC(-/-) cells to apoptosis induced by IFN-gamma and dsRNA. Conversely, inhibition of PKR function by enforced expression of a dominant-negative inhibitory mutant of PKR (PKRDelta6) substantially reduced the IFN and dsRNA hypersensitivity of FANCC(-/-) cells. Two PKR target molecules, IkappaB-alpha and IRF-1, were not differentially activated in FANCC(-/-) cells, but enforced expression of a nonphosphorylatable form of eukaryotic translation initiation factor-2alpha reversed the PKR-mediated block of messenger RNA translation and partially abrogated the PKR-mediated apoptosis in FANCC(-/-) cells. Because no evidence was found of a PKR/FANCC complex in normal cells, it was concluded that an essential function of FANCC is to suppress, indirectly, the activity of PKR and that FANCC inactivation results in IFN hypersensitivity, at least in part, because this function of FANCC is abrogated.

Interferon-gamma-induced apoptotic responses of Fanconi anemia group C hematopoietic progenitor cells involve caspase 8-dependent activation of caspase 3 family members.

Hematopoietic progenitor cells (HPC) from mice nullizygous at the Fanconi anemia (FA) group C locus and children with Fanconi anemia group C (FA-C) are hypersensitive to interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha. This hypersensitivity results, in part, from the capacity of these cytokines to prime the fas pathway. Because fas-mediated programmed cell death in many cells involves sequential activation of specific caspases, we tested the hypothesis that programmed cell death in FA HPC involves the ordered activation of specific caspase molecules. Lysates from lymphoblasts treated with both agonistic anti-fas antibody and IFN-gamma contained activated caspase 3 family members (caspases 3, 6, and 7), as well as caspase 8, whereas activation of caspases 1, 2, 4, 9, and 10 was not detected. The apoptotic effects of fas agonists in IFN-gamma-treated human and murine FA-C cells were blocked when pretreated with inhibitors (ac-DEVD-cho, CP-DEVD-cho, Z-DEVD-FMK) of the caspase 3 protease. Inhibitors (ac-YVAD-cho, CP-YVAD-cho, Z-YVAD-FMK) of caspase 1 did not block apoptosis or caspase 3 activation. Treatment of FA cells with the fluoromethyl ketone tetrapeptide caspase 8 inhibitor (ac-IETD-FMK) did suppress caspase 3 activation. A 4-fold greater fraction of IFN-induced FA-C cells expressed caspase 3 than FA-C cells complemented by retroviral-mediated transfer of FANCC. Therefore fas-induced apoptosis in Fanconi anemia cells of the C type involves the activation of caspase 8, which controls activation of caspase 3 family members and one direct or indirect function of the FANCC protein is to suppress apoptotic responses to IFN-gamma upstream of caspase 3 activation. (Blood. 2000;96:4204-4211)

The Fanconi anemia group C gene product modulates apoptotic responses to tumor necrosis factor-alpha and Fas ligand but does not suppress expression of receptors of the tumor necrosis factor receptor superfamily.

Exposure of hematopoietic progenitor cells (HPC) from mice and humans with Fanconi anemia group C (FAC) to interferon-gamma (IFN-gamma) or tumor necrosis factor-alpha (TNF-alpha) at doses too low to inhibit growth of normal HPC induces profound apoptotic responses. Because the IFN-gamma hypersensitivity of cells lacking the FAC protein is mediated, in part, through priming of the Fas pathway, and because several other members of this family are capable of inducing apoptosis either alone or in concert with each other, we tested the hypothesis that IFN-gamma induces increased expression of members of the TNF receptor (TNFR) superfamily in cells nullizygous for the FAC gene. Using isogenic human Epstein-Barr virus-transformed lymphoblast cell lines and c-kit+ bone marrow cells from mice with inactivating mutations of the FAC locus, we quantified mRNA levels by reverse transcriptase polymerase chain reaction and surface expression of the gene products by flow cytometry of TNFR1, TNFR2, Fas, CD30, CD40, and nerve growth factor receptor. We found that neither constitutive nor IFN-gamma-induced expression of these receptors was influenced by the absence of a functional FAC gene product, and expression of these receptors was not suppressed in nullizygous cells complemented with the normal FAC cDNA. We conclude that, although exaggerated apoptotic responses in FAC-deficient cells are at least partially mediated through activation of members of the TNFR superfamily, the normal FAC protein does not function as a direct suppressor of this family of molecules and inactivation of FAC does not augment expression of these proteins.

Expression of the Fanconi anemia group C gene in hematopoietic cells is not influenced by oxidative stress, cross-linking agents, radiation, heat, or mitotic inhibitory factors.

The Fanconi anemia group C gene (FAC) encodes a 63-kDa protein that plays a role in the growth and differentiation of hematopoietic progenitor cells and in cellular resistance to bifunctional cross-linking agents. The function of the gene product is unknown, as are the factors that govern expression of the gene itself. Seeking to associate a function of this protein with a general metabolic pathway, we attempted to identify factors that induce or repress expression of the gene encoding it. Using two plasmids from which mutant FAC mRNA molecules were transcribed in vitro to serve as competitor mRNAs in quantitative-competitive reverse transcriptase-polymerase chain reaction analysis and novel rabbit antisera raised to recombinant FAC proteins, we quantified gene expression in human hematopoietic cells. We determined that FAC is expressed constitutively in unstimulated normal peripheral blood mononuclear leukocytes, in Epstein-Barr virus (EBV)-transformed B lymphocytes, and in the factor-dependent human myeloid leukemic cell line MO7e at levels of approximately 2000, 200, and 200 FAC mRNA molecules/cell, respectively, and in CD34+ cells from normal human bone marrow at approximately 2000 FAC mRNA molecules/cell. Neither mRNA nor protein increased in any of the cells studied after exposure to mitomycin C, diepoxybutane, hydrogen peroxide, gamma radiation, heat, transforming growth factor-beta, or interferon-gamma. Using these sensitive methods, we confirmed that the FAC gene is constitutively expressed, even in the face of extracellular factors for which the gene product is a known effector of resistance. We conclude that the protective functions of the FAC gene product do not depend upon stressor-induced FAC gene expression.

Inactivation of the Fanconi anemia group C gene augments interferon-gamma-induced apoptotic responses in hematopoietic cells.

Hematopoietic progenitor cells (HPC) from mice nullizygous at the Fanconi anemia (FA) group C locus (FAC -/-) are hypersensitive to the mitotic inhibitory effects of interferon (IFN-gamma). We tested the hypothesis that HPC from the bone marrow of Fanconi group C children are similarly hypersensitive and that the fas pathway is involved in affecting programmed cell death in response to low doses of IFN-gamma. In normal human and murine HPC, IFN-gamma primed the fas pathway and induced both fas and interferon response factor-1 (IRF-1) gene expression. These IFN-gamma-induced apoptotic responses in HPC from the marrow of a child with FA of the C group (FA-C) and in FAC -/- mice occurred at significantly lower IFN doses (by an order of magnitude) than did the apoptotic responses of normal HPC. Treatment of FA-C CD34+ cells with low doses of recombinant IFN-gamma, inhibited growth of colony forming unit granulocyte-macrophage and burst-forming unit erythroid, while treatment with blocking antibodies to fas augmented clonal growth and abrogated the clonal inhibitory effect of IFN-gamma. Transfer of the normal FAC gene into FA-C B-cell lines prevented mitomycin C-induced apoptosis, but did not suppress fas expression or inhibit the primed fas pathway. However, the kinetics of Stat1-phosphate decay in IFN-gamma-treated cells was prolonged in mutant cells and was normalized by transduction of the normal FAC gene. Therefore, the normal FAC protein serves, in part, to modulate IFN-gamma signals. HPC bearing inactivating mutations of FAC fail to normally modulate IFN-gamma signals and, as a result, undergo apoptosis executed through the fas pathway.