Fibroblast growth factor receptor 1 gene expression is required for cardiomyocyte proliferation and is repressed by Sp3.

Fibroblast growth factor receptor 1 (FGFR1) is the only high-affinity FGFR in the vertebrate myocardium. FGFR1 is a tyrosine kinase receptor and has a non-redundant role in proliferation and differentiation of cardiomyocytes during embryogenesis. Results presented here demonstrate that FGFR1 gene expression declines as neonatal cardiomyocytes develop into adult cardiomyocytes. Furthermore, silencing FGFR1 gene expression reduced neonatal cardiomyocyte proliferation, indicating that FGFR1 gene expression is required for the optimal proliferative capacity of cardiomyocytes. To determine the mechanism that governs FGFR1 gene expression in cardiomyocytes, sequence analysis of the proximal mouse FGFR1 promoter identified a potential binding site for Sp transcription factors. Mutation of this site increased FGFR1 promoter activity compared to the wild-type promoter, indicating the presence of a negative transcriptional regulator of the FGFR1 promoter at this site in cardiomyocytes. Sp3 expression in neonatal cardiomyocytes and Drosophila SL2 cells reduced FGFR1 promoter activity in a dose-dependent manner. Western blots and immunocytochemistry indicated that Sp3 was present in the nuclear and cytoplasmic compartments of neonatal cardiomyocytes. Chromatin-immunoprecipitation studies verified that endogenous Sp3 in cardiomyocytes interacts with the FGFR1 promoter. Transient chromatin-immunoprecipitation studies using wild-type and mutated FGFR1 promoter constructs in SL2 cells identified the specific Sp3 binding site within the FGFR1 promoter. These studies implicate Sp3 as a negative transcriptional regulator of FGFR1 promoter activity in cardiomyocytes and as a suppressor of cardiomyocyte proliferation.

Sp1 is required for transcriptional activation of the fibroblast growth factor receptor 1 gene in neonatal cardiomyocytes.

Fibroblast growth factor receptor 1 (FGFR1) is the predominant FGFR in cardiac tissue and regulates proliferation, differentiation, and maintenance of normal myocardium. During development of cardiac tissue, FGFR1 gene expression regulates cardiomyocyte proliferation. The focus of this study was to determine the molecular mechanism of transcriptional activation of the FGFR1 gene in proliferating neonatal cardiomyocytes. Analysis of DNA sequence of the FGFR1 gene identified three potential Sp factor binding sites located at 49 bp, 68 bp, and 100 bp upstream from the 3' end of the promoter segment. Mutation of each of these sites resulted in a significant decline in FGFR1 promoter activity compared to wild type promoter activity, and combinatorial mutation of all three sites completely abrogated promoter activity to background levels. In addition, overexpression of Sp1 in neonatal cardiomyocytes resulted in a dose-dependent increase in wild type FGFR1 promoter activity. However, Sp1-mediated up-regulation of promoter activity was abrogated when all three Sp interacting sites were mutated. Chromatin immunoprecipitation (ChIP) assays were used to demonstrate direct interactions of Sp1 with the proximal promoter region of the FGFR1 gene in neonatal cardiomyocytes. ChIP assays using Drosophila Schneider Line 2 (SL2) cells transiently transfected with wild type or mutant FGFR1 promoter constructs verified the direct interaction between Sp1 and the three Sp1 interacting sites of the promoter. Western blot analyses indicated that Sp1 was present in cytoplasmic and nuclear extracts of neonatal myocardium. These results indicate that Sp1 is a necessary positive regulator of FGFR1 gene transcription in neonatal cardiomyocytes.

Children in need of Pharmacare: medication funding requests at the Toronto Hospital for Sick Children.

OBJECTIVES: Although a national Pharmacare program ensuring access to and affordability of needed medications has repeatedly been cited as a priority to policymakers, 20% of families remain either uninsured or under-insured. The Hospital for Sick Children's Patient Amenities Fund (PAF) covers out-of-pocket medication expenses for inpatient and outpatient children. The research objectives were to 1) examine family demographics and socio-economic status (SES), the types of medications requested and government program process issues of PAF applicants in 1998 and 1999, and 2) describe trends in PAF requests from 1998 to 2000. METHODS: Data were extracted retrospectively from fund requests, charts and social work and discharge planning reports. Descriptive statistics were used to summarize the data and to examine time trends. RESULTS: Eighty-six applicants submitted 112 requests from 1998-1999. Most were for children with cancer, neurological disorders and transplant patients. Medication expenditures were 22,408 dollars in 1999, a 39% increase over 1998. Most requests came from two-parent nuclear families where one or both parents were employed. High deductibles, waiting time, application form complexity and request denials were cited as problems encountered with government drug plans. DISCUSSION: The findings suggest that for provinces that do not provide universal drug insurance programs, relying on a patchwork of government plans and community agencies may not be effective in ensuring easy and timely access to necessary medications for children.