New and evolving rare diseases research programs at the National Institutes of Health.

Research emphasis on rare diseases and orphan products remains a major focus of the research Institutes and Centers of National Institutes of Health (NIH). NIH provides more than USD 31 billion annually in biomedical research and research support. This research is the basis of many of the health advances in rare and common diseases. Numerous efforts and a major emphasis by the public and private sector initiatives have resulted in an increase of interventions and diagnostics for rare diseases. Newer translational research programs provide a more systematic and coordinated approach to rare diseases research and orphan products development. The approach that is offered requires extensive public-private partnerships with the pharmaceutical industry, contract research organizations, philanthropic foundations, medical and scientific advisory boards, patient advocacy groups, the academic research community, research and regulatory scientists, government funding agencies, and the public. Each program is unique and requires lengthy planning and collaborative efforts to reach programmatic goals.

Cbl-b-dependent coordinated degradation of the epidermal growth factor receptor signaling complex.

Cbl proteins function as ubiquitin protein ligases for the activated epidermal growth factor receptor and, thus, negatively regulate its activity. Here we show that Cbl-b is ubiquitinated and degraded upon activation of the receptor. Epidermal growth factor (EGF)-induced Cbl-b degradation requires intact RING finger and tyrosine kinase binding domains and requires binding of the Cbl-b protein to the activated EGF receptor (EGFR). Degradation of both the EGFR and the Cbl-b protein is blocked by lysosomal and proteasomal inhibitors. Other components of the EGFR-signaling complex (i.e. Grb2 and Shc) are also degraded in an EGF-induced Cbl-b-dependent fashion. Our results suggest that the ubiquitin protein ligase function of Cbl-b is regulated by coordinated degradation of the Cbl-b protein along with its substrate. Furthermore, the data demonstrate that Cbl-b mediates degradation of multiple proteins in the EGFR-signaling complex.

Loss of interferon alpha and interferon tau-induced antiviral protection in interferon regulatory factor-2 DNA-binding domain dominant negative mutants.

The role of the interferon regulatory factory (IRF) family of transcription factors in regulation of interferon alpha and interferon tau antiviral activity was investigated using a dominant negative mutant of IRF-2. The IRF-2 DNA binding domain (DBD), without the C-terminal regulatory region, was stably transfected into myeloid U937 cells. Expression of the IRF-2 DBD resulted in an increase in constitutive 2'5' oligoadenylate synthetase (OAS) levels, indicative of an active repressive mechanism, but was not sufficient to protect cells from challenge with vesicular stomatitis virus. Treatment of the DBD clones with interferons alpha A and tau failed to upregulate 2'5' OAS expression and did not elicit an antiviral response. While interferon alpha A was more sensitive than interferon tau to the inhibitory effects of the IRF-2 DBD, IRF-mediated gene induction is involved in successful interferon alpha and tau-induced anti-VSV activity.

An IFN regulatory factor-2 DNA-binding domain dominant negative mutant exhibits altered cell growth and gene expression.

In order to study interferon regulatory factor (IRF) family mediation of cell growth regulation, we established U937 cell lines stably transfected with a truncated form of IRF-2 lacking the transcriptional repressor domain. The truncated IRF-2 contained the DNA binding domain (DBD) and bound the ISRE. Phenotypically, the IRF-2 DBD transfectants exhibited reduced cell growth, altered morphology and increased cell death. Consistent with alterations in growth characteristics, the IRF-2 DBD transfectants constitutively expressed higher levels of the cyclin dependent kinase inhibitor p21WAF1/Cip1 than did control clones. The level of p21WAF1/Cip1 expression was positively correlated with the level of DBD expressed, as well as with the level of growth inhibition in these clones. DBD expression also correlated with expression of other members of the growth regulatory complex, cyclin dependent kinase 2 and cyclin A, but not proliferating cell nuclear antigen. These results imply active repression by IRF-2 to keep p21WAF1/Cip1 transcriptionally silent.

cbl-b inhibits EGF-receptor-induced apoptosis by enhancing ubiquitination and degradation of activated receptors.

Studies in C. elegans and Drosophila melanogaster suggest that cbl proteins are inhibitors of epidermal growth factor receptor (EGFR) function. Here we describe that overexpression of cbl-b, a homologue of the c-cbl protooncogene, inhibits EGFR-induced apoptosis in MDA-MB-468 breast cancer cells. Overexpression of cbl-b results in a shortened duration of EGFR activation upon EGF stimulation. This is demonstrated by decreased amounts of phosphorylated EGFR as well as by inhibition of multiple downstream signaling pathways. The inhibition of signaling by cbl-b results from increased ubiquitination and degradation of the activated EGFR. The inhibitory effects of cbl-b overexpression on apoptosis and on EGFR signaling are reversed by blocking proteosomal degradation of the EGFR. These data demonstrate that the mechanism by which cbl-b inhibits EGFR-induced apoptosis is by activation-dependent degradation of the EGFR. They imply that this mechanism may be a general one whereby cbl proteins regulate intracellular signaling.

Interferon regulatory factor-1 is a major regulator of epidermal growth factor receptor gene expression.

Overexpression of the epidermal growth factor receptor (EGFR) occurs in many tumors and in breast cancer correlates with poor prognosis for treatment. Here, we report that interferon regulatory factor-1 (IRF-1) induces EGFR promoter activity up to 200-fold compared to 3-10-fold induction by other regulators. The region of the promoter that is required for this induction was defined using deletion mutants. In addition, we found that IRF-1 and tricostatin A, a deacetylase inhibitor, have a synergistic effect on EGFR promoter activity. This indicates that the increase in EGFR promoter activity by IRF-1 may also involve changes in chromatin structure. These results identify IRF-1 as a major regulator of EGFR gene expression.