FGF10 induces activation of canonical WNT signaling in the developing mouse liver

ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS during the hepatocyte repair response to liver injury is through HNF-6 effect on cyclin D1.

3. CHALLENGES CONFRONTING FEMALE SURGICAL LEADERS: OVERCOMING THE BARRIERS EDUCATION. Kass RB, Souba WW, Thorndyke LE; Penn State College of Medicine Introduction: Despite increases in the number of female physicians, the surgical specialties continue to attract relatively few women. The absolute number of women reaching the top ranks in academic surgery is remarkably low. The purpose of this study was to: 1) identify barriers/ obstacles to becoming a top female surgical leader; 2) identify key attributes and practices that enable advancement and success, and; 3) identify current challenges in their senior leadership roles. Methods: Semi-structured interviews of ten senior female surgical leaders (departmental chair, center director, surgeon-in-chief) were organized around the following dimensions: personal attributes for success, lessons learned, mistakes to avoid, key career steps, the role of mentoring, gender advantages/disadvantages, and challenges faced as senior leaders. Results: Perseverance (60%) and drive (50%) were identified as critical success factors, as were good communication skills, a passion for scholarship, a stable home life and a positive outlook. 80% percent identified discrimination or gender prejudice as a major obstacle in their careers. Hostile environments and limited job opportunities were noted as other barriers. While 90% percent had mentors and 80% had multiple mentors, 50% of top female academic surgeons acknowledged that they had not been effectively mentored. Key career advice included the following: Develop broad career goals (50%); select a conducive environment (30%); find an empowered mentor (60%); take personal responsibility (40%); organize time and achieve life balance (40%); network (30%); create a niche (30%); pursue research (30%); publish (50%); speak in public (30%) and enjoy the process (30%). Being in a minority, being highly visible and being more collaborative were identified as advantages for female surgeons. Obtaining buy-in and achieving consensus was the greatest on-going leadership challenge reported by 60% of those interviewed. Conclusions: Female academic surgeons face unique challenges to their career advancement. While these barriers (male dominated cultures, gender discrimination, limited job opportunities) are real, they can be overcome by resolve, commitment, and developing strong communication skills. These elements should be taken into consideration in designing career development programs for junior female surgical faculty. 4. IDENTIFICATION OF A NOVEL GATA BINDING ACTIVITY OF CYCLIN D1 PROMOTER DURING EPIDERMAL GROWTH FACTOR (EGF)-INDUCED HEPATOCYTE PROLIFERATION AND AFTER PARTIAL HEPATECTOMY. Hernandez-Garcia A, Thevananther S, Awad SS; Baylor College of Medicine Background: GATA factors contain zinc finger motifs and act by binding to the consensus DNA sequence (A/T) GATA (A/G). GATA factors have been shown to play critical roles in gene regulation of cell proliferation through the interaction with other transcription

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factors such as serum response factor (SRF). Following 70% partial hepatectomy (PH) in the rat, we have observed increased GATA 4 and SRF nuclear protein expression at 24 hours, the known peak of DNA synthesis. We have previously reported increased SRF binding to the Cyclin D1 promoter in hepatocytes stimulated to proliferate with EGF and in regenerating liver after PH. Examination of the 5’ flanking region of the Cyclin D1 promoter, which contains the SRF DNA binding site, revealed possible GATA 4 binding domain. Given the known interactions of GATA 4 and SRF, the purpose of this study was to investigate whether GATA 4 also interacts with the Cyclin D1 promoter. Methods: To address the possibility of GATA 4 as a potential mediator of the observed increase in Cyclin D1 expression following EGF stimulation, the regulatory regions of the rat Cyclin D1 promoter were examined and we identified GATA 4 binding elements upstream of the start site. Primary rat hepatocytes were plated at a density of 50,000 cells/cm 2 and placed in serum free medium 3 hours after plating. Hepatocytes were treated with EGF (20 ng/ml) at 42 hours, previously reported as the mitogen restriction point (JBC: 271:11484,1996), and vehicle treated hepatocytes served as controls. To determine the GATA 4 binding activity of the Cyclin D1 promoter, electromobilityshift assay (EMSA) was performed by incubating radiolabeled oligonucleotides representing the GATA 4 element of the Cyclin D1 promoter with the nuclear extracts obtained from the untreated and EGF treated hepatocytes. The specificity of GATA 4 binding was confirmed by pre-incubating nuclear extracts with a 100-fold excess of specific (SP) and non-specific (NSP) cold competitors prior to the binding with labeled oligonucleotides. To determine the GATA 4 binding activity of the Cyclin D1 promoter in vivo, nuclear extracts were prepared at various time points (1,3,6 hours) from the remnant liver lobes following 70% PH in Sprague Dawley rats and EMSA was performed with the GATA 4 oligonucleotides. Nuclear extracts from normal resected liver lobes of each animal served as controls. Additionally, supershift assay was performed using a monoclonal GATA 4 antibody to confirm GATA 4 binding. Results: Our results suggest that EGF treatment of hepatocytes for two hours in culture leads to increased GATA 4 binding activity of the Cyclin D1 promoter. Correspondingly, 70% PH induces GATA 4 binding activity of the Cyclin D1 promoter in the remnant liver (Figure 1). Conclusion: Our findings demonstrate increased GATA 4 binding to a novel GATA 4 binding site in the Cyclin D1 promoter in hepatocytes stimulated to proliferate with EGF and in regenerating liver after PH. Therefore, GATA 4 may be a key transcription factor required for the proliferation of hepatocytes and may be involved in the mitogen dependant phase of hepatocyte proliferation via the activation of the Cyclin D1 promoter. This warrants further investigation. Fig 1. GATA 4 binding activity of Cyclin D1 promoter from nuclear extracts of remnant lobes 3 hours following PH.

5. FGF10 INDUCES ACTIVATION OF CANONICAL WNT SIGNALING IN THE DEVELOPING MOUSE LIVER. Choe A, Veltmaat JM, Sala FG, Del Moral P, De Langhe S, Stein J, Ford H, Warburton D, Bellusci S, Wang KS; Keck School of Medicine Background: The liver has significant regenerative capacity. Growth factors and signaling cascades that regulate organogenesis are commonly involved in organ regeneration. Thus, the study of the developing liver is useful in understanding the mechanisms involved in liver regeneration. We have evidence that Fibroblast Growth

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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS

Factor 10 (FGF10) is involved in both early hepatogenesis and liver regeneration following partial hepatectomy. WNT proteins also play a major role in early hepatogenesis by acting as survival and proliferative factors for liver progenitor cells. We hypothesized that FGF10, acting through its receptor, FGFR2b, activates canonical WNT signaling to promote survival and proliferation of liver progenitor cells during hepatogenesis. Methods: Both loss and gain of function experiments were designed to test this hypothesis. 1) To study the effect of the loss of Fgfr2b expression expression on canonical Wnt signaling, embryonic mouse livers were dissected at embryonic day (E) 11.5 from Fgfr2b ⫺/⫺TOPGAL ⫹/⫺ and Fgfr2b ⫹/ ⫹/⫺ ⫹ TOPGAL strains and stained for ␤-Galactosidase. TOPGAL mice express ␤-Galactosidase as a readout of activated canonical WNT signaling, allowing us to spatially characterize WNT activation. 2) To determine the effect of FGF10-gain of function on canonical WNT activation and cell survival, livers were dissected from TOPGAL ⫹/⫹ mice at E11.5 and cultured 24 hours in Matrigel™ with DMEM-F12 in the absence or presence of 250ng/mL FGF10 at 37°C, 7.5% CO 2. The liver explants were then analyzed for intensity and distribution of ␤-Galactosidase activity. Relative levels of apoptosis were compared using a TUNEL assay for DNA-strand breaks. Results: Fgfr2b ⫺/⫺TOPGAL ⫹/⫺ cultured livers are smaller, asymmetrical, and express less ␤-Galactosidase activity compared to Fgfr2b ⫹/ ⫹/⫺ ⫹ TOPGAL cultured livers. Both control and FGF10-treated TOPGAL livers explants express ␤-Galactosidase activity in a highly specific, ring-like pattern. However, liver explants treated with FGF10 express stronger and broader levels of ␤-Galactosidase, suggesting FGF10-induced activation of the canonical WNT signaling. TUNEL staining revealed markedly decreased apoptosis in livers cultured with FGF10 in comparison to untreated livers. Conclusions: During liver development, FGF10 modulates canonical WNT signaling, possibly through the activation of its receptor, FGFR2b. Such activation leads to decreased apoptosis, suggesting that FGF10 may act as a survival factor via the activation of the canonical Wnt pathway during the critical early stages of liver organogenesis and may play a significant role in liver regeneration. Further studies elucidating the mechanistic nature underlying the relationship between the FGF and WNT signaling pathways is crucial to our understanding of liver regeneration and of the factors that govern both therapeutic and pathologic liver proliferation.

6. PATTERNS OF CARDIAC CARE WITH IMPLICATIONS FOR VOLUME OUTCOME RELATIONSHIPS. Ricciardi R, Dahlberg PS, Baxter NN; University of Minnesota Medical School Introduction: Hospital volume of coronary artery bypass grafting (CABG) surgery is associated with patient outcome, yet trends in the rate of CABG and percutaneous coronary interventions (PCI) are unknown. We sought to evaluate volume trends in CABG and PCI procedures in the US. Methods: We reviewed data from the Nation-

wide Inpatient Sample from 1988 to 2003. Utilizing ICD-9 codes, we recorded all CABG or PCI procedures. We recorded trends while age-adjusting to the 2000 US population. To evaluate the influence of these trends on hospital CABG volume, we determined the proportion of patients treated at “very-low volume” (⬍230cases/yr) and “very-high volume” hospitals (⬎850cases/yr) as well as the proportion of hospitals considered “very-low volume” or “very-high volume”. Results: CABG was performed in 1,082,218 patients and PCI in 1,589,942. The rate of CABG increased from 5.5 cases per 1000 discharges in 1988 to 8.4 cases in 1997 and then declined to 6.1 cases in 2003 (p⬍0.01). PCI rates tripled from 5.5 cases per 1000 discharges in 1988 to 15.3 cases in 2003 (p⬍0.01). The number of patients treated at “very-low volume” hospitals decreased from 18% in 1988 to 9% in 1997 but increased to 21.3% in 2003 (p⬍0.01). The number of patients treated at “very-high volume” hospitals increased from 21.1% in 1988 to 35% in 1997 but declined to 23.2% in 2003 (p⬍0.01). The number of hospitals considered “very-low volume” declined from 51.2% in 1988 to 33% in 1997 and rose to 48% in 2003 (p⬍0.01). The number of hospitals considered “very-high volume” increased from 6.2% in 1988 to 13% in 1997 and returned to 6% in 2003 (p⬍0.05). Conclusion: The total volume of CABG procedures, the number of patients treated at “very-high volume” hospitals, and the proportion of “very-high volume” hospitals has declined significantly, while the rate of PCI has tripled. Given the established volume-outcome relationship for CABG, these findings have potentially important implications for patient outcomes, fellowship training, and regionalization of care.

7. THE ROLE OF THE GROWTH ARREST-SPECIFIC HOMEOBOX GENE GAX IN INHIBITING ANGIOGENESIS AND NUCLEAR FACTOR-KAPPAB SIGNALING. Patel S, Chen Y, Sohail H, Leal AD, Gorski DH; UMDNJ-Robert Wood Johnson Medical School Introduction: The growth and metastasis of tumors depends upon angiogenesis, but much of the transcriptional regulation of endothelial cell (EC) gene expression responsible for the angiogenic phenotype remains to be elucidated. We have previously shown that Gax, a vascular-specific and growth arrest-specific homeobox transcription factor, inhibits EC activation and angiogenesis in vitro and in vivo and that, in microarray expression profiling, Gax upregulates growth arrest-specific genes and downregulates nuclear factor-␬B target genes. We hypothesized that Gax is an important negative transcriptional regulator of the angiogenic phenotype and sought to investigate potential molecular mechanisms by which it inhibits angiogenesis. Methods: We examined Gax expression in ECs during in vitro angiogenesis by tube formation assays using quanitative reverse transcriptase real time polymerase chain reaction (QRTPCR). Next, to confirm this interaction between Gax expression and NF-␬B activity, we performed electrophoretic mobility shift assays (EMSAs) using a consensus NF-␬B binding site and performed cotransfections with a Gax expression vector (pCGN-Gax) and a promoter reporter construct for an NF-␬B-dependent gene, IL-6 (pIL6Luc). Downstream targets were measured by QRT-PCR Finally, we constructed a series of Gax expression plasmids in pCDNA3.1 with targeted deletions of specific domains and tested which domains were necessary for inhibition of NF-␬B activity by Gax. Results: Gax expression was downregulated during tube formation on Matrigel. We also observed that Gax expression strongly inhibited NF-␬B binding to its consensus sequence, as well as inhibiting both basal and TNF-␣-induced IL-6 promoter-dependent Luciferase activity and expression of mRNA for selected NF-␬B targets. Deletion of the C-terminal domain potentiated the ability of Gax to inhibit NF-␬Bdependent promoter activity in ECs, whereas deletion of the homeodomain had little effect on Gax activity. Conclusions: To our knowledge, Gax is the first homeobox gene described that influences NF-␬B-dependent gene transcription in vascular ECs. Because