A novel human keratinocyte-derived autocrine factor (KAF) was purified from conditioned medium by using heparin affinity chromatography as the first step. Purified KAF stimulated the growth of normal human keratinocytes, mouse AKR-2B cells, and a mouse keratinocyte cell line (BALB/MK). Heparin sulfate inhibited KAF mitogenic activity on all cell types tested and inhibited the ability of KAF to compete with epidermal growth factor for cell surface binding. Interestingly, KAF stimulated the growth of BALB/MK cells at high cell density but failed to stimulate these cells at clonal density. Protein microsequencing of the first 20 NH2-terminal amino acid residues of purified KAF revealed identity to the NH2 terminus of human amphiregulin (AR).Northern (RNA) blot analysis with AR-specific cRNA demonstrated that human keratinocytes, as well as mammary epithelial cell cultures, expressed high levels of AR mRNA. In contrast, AR mRNA was not detected in normal human fibroblasts or melanocytes and was present at reduced levels in several mammary tumor cell lines. The mitogenic activity of purified AR was also shown to be inhibited by heparin sulfate, and an AR-specific enzyme-linked immunosorbent assay (ELISA) revealed that KAF and AR are antigenically related. We have previously shown that human keratinocytes can grow in an autocrine manner. Our present study demonstrates that one of the growth factors responsible for this autocrine growth (KAF) is similar or identical to AR and that KAF and AR bioactivity can be negatively regulated by heparin sulfate.

Gamma interferon (IFN-␥) induces transcription of a distinct set of genes by activating STAT1, one member of a family of latent cytoplasmic transcription factors that are activated via phosphorylation on tyrosine residues (6). The IFN-␥ receptor (IFN-␥R) lacks intrinsic tyrosine kinase activity, but on ligand binding, receptor multimerization results in reciprocal activation of JAK1 and JAK2, two Janus tyrosine kinases noncovalently attached to the IFN-␥R ␣ and ␤ chains (2, 20, 22). Phosphorylation of the tyrosine residue on IFN-␥R␣ by JAK molecules creates a docking site for the Src homology 2 domain of STAT1, which is then phosphorylated on tyrosine and ultimately forms a homodimeric transcription factor that translocates to the nucleus (6, 21). The factors that govern the traffic of cytoplasmic STAT molecules to the docking site on the IFN-␥R are unknown. We have recently found that the activation of STAT1 in response to IFN-␥ is suppressed in hematopoietic cells from children with Fanconi anemia of type C (FA-C) and in mice nullizygous at the FA-C locus. However, in the ground state (uninduced by IFN), IFN response factor 1 (IRF-1) is expressed at high levels in mutant FA-C cells (35), suggesting that a non-STAT1 pathway is involved in constitutive activation of IRF-1 in FA cells. In addition, complementation of the defect by retrovirus mediated transfer of normal FANCC cDNA reconstitutes the normal STAT1 response (10,38).Linkage of FANCC function with that of STAT1 provided us with an opportunity to test whether the relationship of these two molecules was direct or indirect. We report herein results of experiments in which the assembly of the fully activated IFN-␥R complex, including STAT1, JAK1, and JAK2, was examined in isogenic murine and human FA-C cells. We report that in IFN-␥-stimulated FA-C cells, phosphorylation of JAK1, JAK2, and IFN-␥R␣ occurs normally, but STAT1 does not dock at the receptor ␣ chain. In FA-C cells nuclear STAT1 is reduced, and IFN fails to induce STAT1-specific DNA-binding complexes and expression of IRF-1. Expression of the normal FANCC cDNA in mutant cells results in normal STAT1 docking and phosphorylation as well as normal induction of nuclear STAT1-DNA complex and normal induction of IRF-1. We also find that a variety of cytokines and hematopoietic growth factors stimulate the association of STAT1 with glutathione S-transferase (GST) fusion proteins encoding the normal FANCC but not a naturally occurring inactivating mutant FANCC (L554P) and that the association occurs rapidly and prior to STAT1 phosphorylation on Y 701 . Coimmunoprecipitation experiments confirmed the IFN-inducible association of

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.

Keratinocytes and fibroblasts isolated from human neonatal foreskin can be plated and grown through multiple rounds of division in vitro under defined serum-free conditions. We utilized these growth conditions to examine the mitogenic potential of acidic and basic fibroblast growth factor (aFGF and bFGF) on these cells. Our results demonstrate that both aFGF and bFGF can stimulate the proliferation of keratinocytes and fibroblasts. aFGF is a more potent mitogen than bFGF for keratinocytes. In contrast, bFGF appears to be more potent than aFGF in stimulating the growth of fibroblast cultures. Heparin sulfate (10 micrograms/ml) dramatically inhibited the ability of bFGF to stimulate the proliferation of keratinocytes. In comparison, heparin slightly inhibited the stimulatory effect of aFGF and had no effect on epidermal growth factor (EGF) stimulation in keratinocyte cultures. In fibroblast cultures the addition of heparin enhanced the mitogenic effect of aFGF, had a minimal stimulatory effect on the mitogenic activity of bFGF, and had no effect on EGF-stimulated growth. Our results demonstrate that the proliferation in vitro of two normal cell types found in the skin can be influenced by aFGF and bFGF and demonstrate cell-type specific differences in the responsiveness of fibroblasts and keratinocytes to these growth factors and heparin.

Tumor necrosis factor alpha (TNF-␣) production is abnormally high in IntroductionThe Fanconi anemia (FA) proteins play an important role in regulating genome stability, 1 but there is little evidence that the loss of the genoprotection per se in FA cells accounts for the molecular pathogenesis of the bone-marrow failure characteristic of this disease. In fact there is evidence that at least some of these proteins are multifunctional 2 and participate in canonical signaling pathways in hematopoietic cells. [2][3][4][5][6][7][8] Fanconi anemia, complementation group C (FANCC)-deficient cells, for example, are hypersensitive to the apoptotic effects of tumor necrosis factor-␣ (TNF-␣). [4][5][6][7][8][9] In addition, FA cells overproduce TNF-␣ for reasons that have not yet been fully explained. [10][11][12] Most importantly, there is clear evidence that overproduction of and hypersensitivity to TNF-␣ in hematopoietic cells of Fancc Ϫ/Ϫ mice results in bone marrow hypoplasia 13,14 and that long-term ex vivo exposure of murine Fancc Ϫ/Ϫ hematopoietic cells to both growth factors and TNF-␣ results in the evolution of cytogenetically marked preleukemic clones. 9 Therefore, the hematopoietic phenotype of FA may evolve from the overproduction of precisely the cytokine to which FA stem cells are hypersensitive. We designed gene expression microarray experiments by using marrow cells from both patients with FA and normal volunteers in part to seek potential clues to the mechanisms by which FA cells overproduce TNF-␣.Recognizing that transcriptomal analysis would not reveal aspects of the FA phenotype that were controlled translationally or posttranslationally, we also conducted a proteomics analysis. We based our experimental design on an accepted function of the FA "nuclear core complex," that is, its capacity to facilitate monoubiquitinylation of both Fanconi anemia, complementation group I and Fanconi anemia, complementation group D2 (FANCD2). 15,16 Although it is clear that monoubiquitinylation, at least of FANCD2, is required for the avoidance of genotoxicity, 17 it seemed to us unlikely that 8 individual FA genes encoding the "core complex proteins" should have evolved to control the monoubiquitinylation of merely 1 or 2 nuclear proteins. Therefore, reasoning that ubiquitinylation of a variety of other proteins might also be influenced by the core FA proteins, we designed a proteomics survey of ubiquitinylated proteins in FA-C cells and isogenic controls. We reasoned that this approach might lead to the identification of other proteins underubiquitinylated in mutant cells. As reported herein, the gene expression microarray analysis revealed a significant overrepresentation of overexpressed ubiquitin pathway genes in the mutant cells. We therefore took into account the alternative possibility that some ubiquitinylated proteins might be found uniquely in the mutant cells.Indeed, one such protein, Toll-like receptor 8 (TLR8), did appear in the ubiquitin-positive fractions only in FANCC-mutant cells. Given that TLR8 activ...