Affirming NIH's commitment to addressing structural racism in the biomedical research enterprise.

NIH has acknowledged and committed to ending structural racism. The framework for NIH's approach, summarized here, includes understanding barriers; developing robust health disparities/equity research; improving its internal culture; being transparent and accountable; and changing the extramural ecosystem so that diversity, equity, and inclusion are reflected in funded research and the biomedical workforce.

The transcription factor GCF2 is an upstream repressor of the small GTPAse RhoA, regulating membrane protein trafficking, sensitivity to doxorubicin, and resistance to cisplatin.

Our aim was to explore the involvement of the transcriptional suppressor GCF2 in silencing RhoA, disorganization of the cytoskeleton, mislocalization of MRP1, and sensitivity to anticancer agents as an upstream gene target in cancer therapy. Increased expression of GCF2 was found in human cisplatin-resistant cells, and overexpression in GCF2-transfected cells results in loss of RhoA expression and disruption of the actin/filamin network. In consequence, the membrane transporter MRP1 was internalized from the cell surface into the cytoplasm, rendering cells sensitive to doxorubicin by more than 10-fold due to increased accumulation of doxorubicin in the cells. The GCF2 transfectants also showed reduced accumulation of cisplatin and increased resistance. siRNA targeted to GCF2 suppressed the expression of GCF2 in cisplatin-resistant cells, reactivated RhoA expression, and restored the fine structure of actin microfilaments. MRP1 was also relocated to the cell surface. siRNA targeted to RhoA increased resistance 3-fold in KB-3-1 and KB-CP.5 cells. These data for the first time demonstrate a novel complex regulatory pathway downstream from GCF2 involving the small GTPase RhoA, actin/filamin dynamics, and membrane protein trafficking. This pathway mediates diverse responses to cytotoxic compounds, and also provides a molecular basis for further investigation into the pleiotropic resistance mechanism at play in cisplatin-resistant cells.

The Imperative to Reimagine Assisted Living.

Assisted living (AL) has existed in the United States for decades, evolving in response to older adults' need for supportive care and distaste for nursing homes and older models of congregate care. AL is state-regulated, provides at least 2 meals a day, around-the-clock supervision, and help with personal care, but is not licensed as a nursing home. The key constructs of AL as originally conceived were to provide person-centered care and promote quality of life through supportive and responsive services to meet scheduled and unscheduled needs for assistance, an operating philosophy emphasizing resident choice, and a residential environment with homelike features. As AL has expanded to constitute half of all long-term care beds, the increasing involvement of the real estate, hospitality, and health care sectors has raised concerns about the variability of AL, the quality of AL, and standards for AL. Although the intent to promote person-centered care and quality of life has remained, those key constructs have become mired under tensions related to models of AL, regulation, financing, resident acuity, and the workforce. These tensions have resulted in a model of care that is not as intended, and which must be reimagined if it is to be an affordable care option truly providing quality, person-centered care in a suitable environment. Toward that end, 25 stakeholders representing diverse perspectives conferred during 2 half-day retreats to identify the key tensions in AL and discuss potential solutions. This article presents the background regarding those tensions, as well as potential solutions that have been borne out, paving the path to a better future of assisted living.

GC-binding factor 2 interacts with dishevelled and regulates Wnt signaling pathways in human carcinoma cell lines.

GC-binding factor 2 (GCF2), a transcriptional repressor that decreases the activity of several genes is capable of binding directly to the GC-rich sequence of the EGFR promoter and repressing the transcriptional activity of EGFR. In addition to its function as a transcriptional repressor, GCF2 can directly interact with other proteins such as flightless-1 (Fli-1). Many previous findings pertaining to the function of Fli-1 have suggested a role for fli-1 in providing a direct link between molecules involved in signal transduction pathways and the actin cytoskeleton. We hypothesized that GCF2, together with Fli-1, plays a role in regulating cytoskeleton function, cell migration, and/or morphology. In our study, we observed that GCF2 is crucial for the activation of RhoA, a small GTPase that plays a key role in the regulation of the actin cytoskeleton. RhoA was markedly inactivated as a result of the decreased expression of GCF2. Co-immunoprecipitations were subsequently performed to further investigate the mechanism for the repressive function. We identified dishevelled (Dvl), which is the key mediator for the Wnt pathway, as a binding partner with GCF2. These results strongly suggest that GCF2 plays a role in the Wnt-noncanonical planar cell polarity (PCP) signaling pathway. Consequently, GCF2 may regulate the cytoskeleton or migration via Dvls and RhoA.

Early Growth Response-1 gene mediates up-regulation of epidermal growth factor receptor expression during hypoxia.

Hypoxia occurs during development of cancers and is correlated with cancer progression. Hypoxia also induces epidermal growth factor receptor (EGFR) expression. The EGFR plays a vital role in cell growth, and its overexpression can lead to transformation. We sought to determine the regulator(s) of EGFR expression during hypoxia. We demonstrate that early growth response factor 1 (Egr-1), which is induced by hypoxia, can activate the basal transcriptional activity of the EGFR promoter. Egr-1 not only transactivates the EGFR promoter activity but also enhances endogenous EGFR expression. Using a series of EGFR promoter deletion mutants, we show that the region between -484 and -389, which contains a putative Egr-1 consensus motif, is crucial for EGFR transactivation by Egr-1. Electrophoretic mobility shift assays show that Egr-1 binds to the oligonucleotide containing this Egr-1 motif. Also, introduction of an antisense oligonucleotide for Egr-1 diminishes EGFR expression during hypoxia, indicating that the up-regulation of EGFR by hypoxia is mediated through Egr-1. Our results provide evidence that regulation of EGFR promoter activity by Egr-1 represents a mechanism for epidermal cell growth during hypoxia.

GCF2: expression and molecular analysis of repression.

GC-binding factor 2 (GCF2) is a transcriptional repressor that decreases activity of the epidermal growth factor receptor (EGFR) and other genes. We have mapped the gene for GCF2 by fluorescence in situ hybridization (FISH) to chromosome 2q37. Sequence analysis of the GCF2 gene and cDNA showed that the gene consists of eight exons and introns and spans 73 kbp of DNA. Northern blot analysis showed that GCF2 mRNA was differentially expressed in many human tissues and cell lines. GCF2 mRNA was expressed as a 4.2 kb mRNA in most human tissues with the highest expression level in peripheral blood leukocytes and lowest expression in brain and testis. Additional transcripts of 6.6, 2.9 and 2.4 kb were found in some tissues but the only transcript detected in cancer cell lines was 4.2 kb with high levels found in seven Burkitts' lymphoma cell lines. Western blot analysis showed that GCF2 protein is present at high levels in Burkitts' lymphoma and several other cancer cell lines. GCF2 was found in both nuclear and cytoplasmic compartments in cells. Deletion mutants of GCF2 revealed that amino acids 429-528 are required for both DNA binding and repression of the EGFR promoter. Furthermore, GCF2 was able to substantially decrease activator protein 2 (AP2) enhancement of the EGFR promoter. Thus, GCF2 is a transcriptional repressor overexpressed in cancer cell lines with a role in regulating expression of the EGFR.

Analysis of the epidermal growth factor receptor promoter: the effect of nuclear factor-kappaB.

The epidermal growth factor receptor gene is highly regulated and responsive to extracellular stimuli that control cell growth. We have identified five putative nuclear factor-kappaB (NF-kappaB) binding sites within the epidermal growth factor receptor (EGFR) promoter region by sequence analysis. We have analyzed the potential role of NF-kappaB family members in the regulation of the EGFR transcription. Electrophoretic mobility shift analysis demonstrated that the p50 and p49, subunit proteins of the NF-kappaB, bound to the EGFR promoter at four out of five of these sites. However, it was found that NF-kappaB could not transactivate the EGFR by cotransfection experiments with each NF-kappaB subunit, using p50, p65 and c-Rel and an EGFR promoter luciferase reporter. Treatment of cells with tumor necrosis factor (TNF)-alpha, which could degrade the I-kappaB and then result in translocation of NF-kappaB to nucleus, did not enhance EGFR promoter reporter gene transcription. Also, TNF-alpha did not induce EGFR expression at the protein level. These results indicate that even though purified NF-kappaB can bind to the putative sites, there is no evidence that NF-kappaB transactivates the EGFR promoter region.

Leucine-rich repeat (in Flightless I) interacting protein-1 regulates a rapid type I interferon response.

The cell autonomous response to viral infection is carefully regulated to induce type I interferons (IFNs), which in turn induce the establishment of an antiviral state. Leucine-rich repeat (in Flightless I) interacting protein-1 (LRRFIP1) and LRRFIP2 are 2 related proteins that have been identified as interacting with MyD88 and Flightless I homolog, a leucine-rich repeat protein. LRRFIP2 positively regulates NFκB and macrophage cytokine production after lipopolysaccharide, but less is known about LRRFIP1. We hypothesized that LRRFIP1 could be more important in antiviral responses, as overexpression led to type I IFN production in a pilot study. The induction of type I IFNs occurred even in the absence of virus, but was enhanced by the presence of virus. Conversely, knockdown of LRRFIP1 compromised IFN expression. We found that LRRFIP1 was rapidly recruited to influenza-containing early endosomes in a p38-dependent fashion. This was specific for virus-containing endosomes as there was almost no colocalization of LRRFIP1 with early endosomes in the absence of virus. Further, LRRFIP1 was recruited to RNA-containing vesicles. Taken together, these data suggest that LRRFIP1 participates in cell responses to virus at early time points and is important for type I IFN induction.

Transactivation of the EGFR by AP-1 is induced by Helicobacter pylori in gastric cancer.

BACKGROUND: Helicobacter pylori infection of the gastric mucosa is strongly associated with gastritis, peptic ulcer disease, and gastric cancer. However, the mechanisms by which H. pylori causes cancer are currently unknown. Binding of epidermal growth factor (EGF) to its receptor (EGFR) may be important in the development of gastric cancer. This interaction accelerates cell proliferation and migration, and triggers epithelial cell signaling. In this study, we investigated the effects of H. pylori on EGFR- and AP-1-mediated signal transduction pathways in the AGS gastric epithelial cell line and gastric tissue from humans. METHODS: Cells were treated with H. pylori and cell death was examined at a variety of time points using cell viability and trypan blue exclusion dye assay. To investigate the effects on EGFR regulation, AGS cells were transfected with a full-length and truncated EGFR luciferase (luc) reporter. Tissue microarray containing 44 samples of gastric biopsies from H. pylori-positive patients was analyzed for protein expression level of EGFR by immunohistochemistry. RESULTS: EGFR promoter activity was increased (twofold) 3 h after treatment with H. pylori commenced. Using a series of EGFR promoter deletion mutants, we identified a region that was crucial for transactivation of the EGFR by H. pylori. To determine whether AP-1 binding was altered, we transfected AGS cells with an AP-1 luciferase construct and then treated them with H. pylori for up to 6 h. We found that AP-1 activity was induced by H. pylori in gastric cells, while electrophoretic mobility shift assays confirmed that binding of AP-1 to the EGFR promoter site was increased following H. pylori treatment. Binding of c-Jun and c-Fos to the EGFR promoter region -1,062/-900 was induced eight- and six fold, respectively, using ChIP assay. Active EGFR staining was markedly increased in gastric mucosa from infected persons, compared to uninfected controls. CONCLUSIONS: We conclude that exposure of gastric cells to H. pylori induces increased production of EGFR through various signal transduction pathways, including those mediated by the EGFR and AP-1. Distinct effects on EGFR activation may specify the subset of AP-1 target genes that are selected, including those involved in proliferation and apoptosis. This is consistent with EGFR activation that was found in the gastric mucosa of humans infected with H. pylori. Hence, the balance between apoptosis and proliferation in these cells may be altered in response to injury caused by H. pylori infection, leading to an increased risk of cancer.