Gregory R. Faulkner

FANCC interacts with Hsp70 to protect hematopoietic cells from IFN-γ/TNF-α-mediated cytotoxicity

The Fanconi anemia (FA) complementation group C gene product (FANCC) functions to protect hematopoietic cells from cytotoxicity induced by interferon‐γ (IFN‐γ), tumor necrosis factor‐α (TNF‐α) 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‐γ/TNF‐α‐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‐γ and TNF‐α.

Expression of apoptosis-related genes in chronic cyclosporine nephrotoxicity in mice

To define the mechanism of cyclosporine (CsA)‐induced apoptosis, we investigated the expression of apoptosis‐related genes in experimental chronic CsA nephrotoxicity. Mice on a low‐salt (0.01%) diet were given vehicle (VH, olive oil, 1 mg/kg/day), or CsA (30 mg/kg/day), and sacrificed at 1 and 4 weeks. Apoptosis was detected with deoxynucleotidyl transferase‐mediated dUTP nick end‐labeling (TUNEL) stain, and the expressions of apoptosis‐related genes were evaluated by reverse transcription‐polymerase chain reaction, immunoblot or immunohistochemistry. The activity of caspase 1 and 3 was also evaluated. The CsA group showed increases in apoptotic cells compared with the VH group (54 ± 41 vs. 3 ± 3, p < 0.05), and the number of apoptotic cells correlated well with interstitial fibrosis scores (r = 0.83, p < 0.01). The CsA group showed a significant increase in Fas‐ligand mRNA (0.20 vs. 0.02 amol/μg total RNA, p < 0.05) and Fas protein expression (146% vs. 95%, p < 0.05), compared with the VH group. The CsA group showed significant increases in ICE mRNA (0.21 vs. 0.03 amol/μg total RNA at 4 weeks, p < 0.05) and CPP32 mRNA (0.18 vs. 0.03 amol/μg total RNA at 4 weeks, p < 0.05), compared with the VH group. The enzymatic activity of ICE (16.6 vs. 7.9 ρmol/μg/ h, p < 0.05) and CPP32 protease (15.6 vs. 2.7 ρmol/μg/ h, p < 0.05) proteases were increased in the CsA group, compared with the VH group. The ratio between bax and bcl‐2 protein increased significantly in the CsA group (5.3‐fold), compared with the VH group. Levels of p53 protein also increased in the CsA group. Immunohistochemical detection of Fas, Fas‐ligand, ICE and CPP32 revealed strong immunoreactivity in renal tubular cells in areas of structural injury. These findings suggest that local activation of the apoptosis‐related genes is associated with CsA‐induced apoptotic cell death.

The Fanconi anemia group C gene product modulates apoptotic responses to tumor necrosis factor-α 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.

Influence of HIV-1 on induced expression of granulopoietic factors: The role of HIV-1 vpr

Abstract IL-1-induced expression of granulopoietic factors, including G-CSF, by human bone marrow stromal cells is suppressed by HIV-1 infection, a phenomenon that may account for the regenerative granulopoietic failure often seen in HIV-1 infected patients. Seeking to identify the HIV-1 gene products that account for stromal cell dysfunction, we used replication incompetent HIV-1 mutants to demonstrate that deletion of vpr abrogates the stromal cell defect. Bone marrow stromal cell cultures were established in primaria dishes containing VEGF and were allowed to grow to near confluence. HIV-1 pseudotyped with VSV-G envelope protein was produced by transient co-transfection of 293T cells with a VSV-G expression vector and HIV-1 proviral plasmids containing: (1) a deletion in the envelope gene (env-) or (2) deletions of both env and vpr. Expression of G-CSF, GM-CSF, and IL-6 by stromal cells expressing the env deleted mutant was reduced by more than 50%, but this inhibitory effect was largely relieved by mutants with deletions of both env and vpr. To confirm the vpr effect, we carried out gain-of-function studies using bone marrow stromal cells transduced with the Moloney based expression vector pSLX-CMV- vpr or vector control pSLX-CMVX. IL-6 and G-CSF expression was reduced in stromal cells expressing vpr. We conclude that blunted responses of HIV-1 infected hematopoietic stromal cells depends, at least in part, upon the expression of HIV-1 vpr and that, in the case of G-CSF and IL-6, vpr is sufficient to account for this effect.

Overexpression of IRF-1 and p21WAF1/CIP1 in fanconi anemia (Fa) Cells correlates with STAT-1 phosphorylation defects but is independent of icsbp deficiency

Abstract Hematopoietic progenitor cells from FA group C (FA-C) patients are hypersensitive to IFNγ. IFNγ-inducible genes known to influence cell survival and/or cell cycle progression (IRF-1, p21 (WAF1/CIP1) and MxA) are constitutively expressed in FA-C cells while levels of the transcriptional repressor ICSBP are reduced. The FA-C protein (FANCC) interacts with Stat1 via a conserved central domain (see abstract by Pang, Q et al). Even conservative mutations in this domain result in decreased Stat1 phosphorylation in response to IFNγ. Because induction of ICSBP by IFNγ is mediated by Stat1 and activation of Stat1 is decreased in some FA cells, we sought to determine if overexpression of IRF-1 and p21 is specifically caused by deficiencies in Stat1 activation and ICSBP expression. Using real time RT-PCR and immunoblot analysis we determined that IRF-1 and p21 are upregulated in FA cells that contain a Stat1 phosphorylation defect but not in FA cells in which Stat1 activation is preserved. To determine if IRF-1 and p21 upregulation is dependent on ICSBP deficiency, we transduced Stat1 defective FA-C lymphoblasts with a retroviral vector expressing ICSBP. Constitutive expression of ICSBP failed to down-regulate IRF-1 or p21 correlates positively with Stat1 phosphorylation defects, but enforced expression of ICSBP does not suppress expression of these genes. We conclude that the capacity of the FANCC protein to facilitate Stat1 activation is linked with its capacity to functionally repress expression of IRF-1 and p21 but involves a mechanism other than ICSBP induction.