Luika A. Timmerman

Role of the NF-ATc transcription factor in morphogenesis of cardiac valves and septum

In lymphocytes, the expression of early immune response genes is regulated by NF-AT transcription factors, which translocate to the nucleus after dephosphorylation by the Ca2+-dependent phosphatase, calcineurin. We report here that mice bearing a disruption in the NF-ATc gene fail to develop normal cardiac valves and septa and die of circulatory failure before day 14.5 of development. NF-ATc is first expressed in the heart at day 7.5, and is restricted to the endocardium, a specialized endothelium that gives rise to the valves and septum. Within the endocardium, specific inductive events appear to activate NF-ATc: it is localized to the nucleus only in endocardial cells that are adjacent to the interface with the cardiac jelly and myocardium, which are thought to give the inductive stimulus to the valve primordia. Treatment of wild-type embryos with FK506, a specific calcineurin inhibitor, prevents nuclear localization of NF-ATc. These data indicate that the Ca2+/calcineurin/NF-ATc signalling pathway is essential for normal cardiac valve and septum morphogenesis; hence, NF-ATc and its regulatory pathways are candidates for genetic defects underlying congenital human heart disease.

Different nuclear signals are activated by the B cell receptor during positive versus negative signaling.

It is not known how immunogenic versus tolerogenic cellular responses are signaled by receptors such as the B cell antigen receptor (BCR). Here we compare BCR signaling in naive cells that respond positively to foreign antigen and self-tolerant cells that respond negatively to self-antigen. In naive cells, foreign antigen triggered a large biphasic calcium response and activated nuclear signals through NF-AT, NF-kappa B, JNK, and ERK/pp90rsk. In tolerant B cells, self-antigen stimulated low calcium oscillations and activated NF-AT and ERK/pp90rsk but not NF-kappa B or JNK. Self-reactive B cells lacking the phosphatase CD45 did not exhibit calcium oscillations or ERK/pp90rsk activation, nor did they repond negatively to self-antigen. These data reveal striking biochemical differences in BCR signaling to the nucleus during positive selection by foreign antigens and negative selection by self-antigens.

The T Cell Activation Factor NF-ATc Positively Regulates HIV-1 Replication and Gene Expression in T Cells

Clinical deterioration in human immunodeficiency virus type 1 (HIV-1) infection is associated with increased levels of viral replication and burden in the peripheral blood and lymphoid organs. T cell activation and ensuing cellular gene activation can be critical for HIV-1 replication. The hypothesis that the nuclear factor of activated T cells (NF-AT) may influence HIV-1 replication is therefore compelling given the tight correlation of HIV-1 transcriptional induction to T cell activation. We report that certain NF-AT(Rel) family members productively bind the kappaB regulatory elements, synergize with NF-kappaB and Tat in transcriptional activation of HIV-1, and enhance HIV-1 replication in T cells. These results link regulatory factors critical to T cell commitment directly to HIV-1 replication.

Glutamine Sensitivity Analysis Identifies the xCT Antiporter as a Common Triple-Negative Breast Tumor Therapeutic Target

A handful of tumor-derived cell lines form the mainstay of cancer therapeutic development, yielding drugs with an impact typically measured as months to disease progression. To develop more effective breast cancer therapeutics and more readily understand their clinical impact, we constructed a functional metabolic portrait of 46 independently derived breast cell lines. Our analysis of glutamine uptake and dependence identified a subset of triple-negative samples that are glutamine auxotrophs. Ambient glutamine indirectly supports environmental cystine acquisition via the xCT antiporter, which is expressed on one-third of triple-negative tumors in vivo. xCT inhibition with the clinically approved anti-inflammatory sulfasalazine decreases tumor growth, revealing a therapeutic target in breast tumors of poorest prognosis and a lead compound for rapid, effective drug development.

Lipoprotein lipase links dietary fat to solid tumor cell proliferation.

Many types of cancer cells require a supply of fatty acids (FA) for growth and survival, and interrupting de novo FA synthesis in model systems causes potent anticancer effects. We hypothesized that, in addition to synthesis, cancer cells may obtain preformed, diet-derived FA by uptake from the bloodstream. This would require hydrolytic release of FA from triglyceride in circulating lipoprotein particles by the secreted enzyme lipoprotein lipase (LPL), and the expression of CD36, the channel for cellular FA uptake. We find that selected breast cancer and sarcoma cells express and secrete active LPL, and all express CD36. We further show that LPL, in the presence of triglyceride-rich lipoproteins, accelerates the growth of these cells. Providing LPL to prostate cancer cells, which express low levels of the enzyme, did not augment growth, but did prevent the cytotoxic effect of FA synthesis inhibition. Moreover, LPL knockdown inhibited HeLa cell growth. In contrast to the cell lines, immunohistochemical analysis confirmed the presence of LPL and CD36 in the majority of breast, liposarcoma, and prostate tumor tissues examined (n = 181). These findings suggest that, in addition to de novo lipogenesis, cancer cells can use LPL and CD36 to acquire FA from the circulation by lipolysis, and this can fuel their growth. Interfering with dietary fat intake, lipolysis, and/or FA uptake will be necessary to target the requirement of cancer cells for FA. Mol Cancer Ther; 10(3); 427–36. ©2011 AACR.

Mechanisms of cell-cycle arrest in Spitz nevi with constitutive activation of the MAP-kinase pathway.

Spitz nevi are benign melanocytic nevi that overlap histopathologically with melanoma. We previously found copy number increases of chromosome 11p frequently paralleled by mutations in the HRAS oncogene mapping to this region. In this study, we explored mechanisms that inhibit proliferation in the presence of HRAS activation. We analyzed MAP-kinase activation using immunohistochemistry for phospho-ERK, cyclin D1, and microphthalmia transcription factor expression in 17 Spitz nevi with and 18 Spitz nevi without 11p copy number increase. We found relatively high levels of phospho-ERK and cyclin D1 expression suggesting MAP-kinase pathway activation in both groups of Spitz nevi. However, Spitz nevi with 11p copy number increases showed significantly higher levels of cyclin D1 expression and lower levels of microphthalmia transcription factor expression suggesting stronger MAP-kinase pathway activation in this group. Contrasting this apparent activation, the proliferation rate as assessed by Mib1 expression was low in both groups. An analysis of cell-cycle inhibitory proteins including p16, p21, and p27 showed that the majority of Spitz nevus cells expressed high levels of p16, with cells of the cases that had increased copy number of 11p expressing significantly higher levels than those of Spitz nevi with normal copy number of 11p. We propose that in benign nevi with constitutive activation of the MAP-kinase pathway, p16 functions as an essential mediator of oncogene-induced senescence preventing progression to melanoma.

Nuclear Factor of Activated T Cells Is Associated with a Mast Cell Interleukin 4 Transcription Complex

Interleukin 4 (IL-4), an immunoregulatory cytokine, is produced only by a subset of activated T cells and cells of the mast cell-basophil lineage. The production of IL-4 by mast cells likely represents a significant source of this protein in local immune-inflammatory responses in the skin, brain, gastrointestinal, and respiratory tracts, in which mast cells are prevalent. In the present study, the cis- and trans-acting elements that control inducible mast cell IL-4 gene transcription were examined and compared with those that function in T cells. We demonstrate that, as in T cells, sequences between bp -87 and -70 are critical for protein association and activation-dependent gene transcription and that this region (termed the activation-responsive element region) is the target of an inducible, cyclosporin A-sensitive, DNA-protein interaction. When assessed by electrophoretic mobility shift assays and UV cross-linking analyses, multiple proteins in both T- and mast cell nuclear extracts associate with the activation-responsive element in vitro, and some of these appear identical. However, distinct proteins are associated with each of the complexes as well. AP-1 family members are unique to the T-cell-stimulation-dependent complex, whereas mast cell complexes contain factors that are reactive with anti-nuclear factor of activated T cells p (NF-ATp) and anti-NF-ATc antibodies but have distinct molecular masses compared with those of T-cell-derived NF-AT. Furthermore, an anti-NF-ATp-reactive factor with a molecular mass of approximately 41 kDa is present in the nuclei of unstimulated cells and binds independently of cell activation, unlike the previously described NF-AT family members. These data support the idea that there are uniquely regulated, cell lineage-specific transcription factors related to T-cell-derived NF-AT that mediate inducible IL-4 transcription in mast cells. These differences likely reflect the distinct cell surface signaling requirements for IL-4 production in T and mast cells.

Molecular characterization of MHC class II antigens (β1 domain) in the BB diabetes-prone and-resistant rat

The BB or BB/Worcester (BB/W) rat is widely recognized as a model for human insulin-dependent diabetes mellitus (IDDM). Of at least three genes implicated in genetic susceptibility to IDDM in this strain, one is clearly linked to the major histocompatibility complex (MHC). In an attempt to define the diabetogenic gene(s) linked to the MHC of the BB rat, cDNA clones encoding the class II MHC gene products of the BB diabetes-prone and diabetes-resistant sublines have been isolated and sequenced. For comparison, theβ1 domain of class II genes of the Lewis rat (RTlL) were sequenced. Analysis of the sequence data reveals that the first domain of RT1.Dβ and RT1.Bβ chain of the BB rat are different from other rat or mouse class 11 sequences. However, these sequences were identical in both the BB diabetes-prone and BB diabetes-resistant sublines. The significance of these findings is discussed in relation to MHC class II sequence data in IDDM patients and in the nonobese diabetic (NOD) mouse strain.

Major histocompatibility complex associations with systemic lupus erythematosus

This study focused on clinical subsets within systemic lupus erythematosus (SLE) in order to identify more homogeneous patient groups in which to define disease susceptibility gene(s). Analysis of the major histocompatibility complex gene products and genes with major histocompatibility complex class II and class III locus-specific probes and oligonucleotide probes for selected human leukocyte antigen DQ-beta alleles showed significant increases of human leukocyte antigen DR2 and the rare DQ-beta allele DR2-DQw1.AZH in the lupus nephritis patients compared with lupus patients without renal disease (relative risk = 8.3). C4A null was detected in one third of all of the SLE patients. In two thirds of the C4A null patients this was due to a DR3-associated C4A gene deletion. The remaining third may have a regulatory defect and this was DR2-associated. DR4 was significantly decreased in the nephritis patients in comparison with the non-renal SLE patients (relative risk = 0.3). A novel DQ-beta gene has been sequenced from two SLE patients that has not been observed in the normal population. Potential implications of these findings are discussed.

Cloning and chromosomal localization of the human and murine genes for the T-cell transcription factors NFATc and NFATp

The nuclear factor of activated T cells (NFAT) is a transcription factor complex involved in the activation of cytokines and cell surface molecules associated with coordinating the actions of different cells required for an immune response. Two different genes have recently been cloned that encode proteins capable of functioning as the pre-existing (p) and cytosolic (c) component of the NFAT transcription complex, NFATc of human and NFATp of murine origin (Northrop et al., 1994; McCaffrey et al., 1993b). We report here the partial cDNA cloning of the murine homolog of NFATc and the human homolog of NFATp, and the chromosomal localization of both genes in both species to conserved syntenic regions. Through the use of mapping panels of human x Chinese hamster and mouse x rodent cells hybrids, the NFATc genes were mapped to human and mouse chromosomes 18. By analyzing a chromosome 18 radiation hybrid panel, the human NFATc gene was localized to the q terminus, closely linked to STS marker D18S497. The murine Nfatc gene was sublocalized to chromosome band 18E4 by FISH. The NFATp genes were mapped by somatic cell hybrid analysis to human chromosome 20 and mouse chromosome 2. Human NFATp was assigned to chromosome region 20q13.2-->q13.3 by FISH. Based on the conserved syntenic region on human chromosome 20 and mouse chromosome 2, murine Nfatp is predicted to reside in the vicinity of a mutant locus wasted. Homozygous wst/wst mice display a phenotype reminiscent of severe combined immune deficiency or ataxia telangiectasia, disorders that could therefore be considered candidates for NFATp mutations.