Genetic elimination of prothrombin in adult mice is not compatible with survival and results in spontaneous hemorrhagic events in both heart and brain.

Mice carrying a conditional prothrombin knockout allele (fII(lox)) were established to develop an experimental setting for exploring the importance of thrombin in the maintenance of vascular integrity, the inflammatory response, and disease processes in adult animals. In the absence of Cre-mediated recombination, homozygous fII(lox/lox) mice or compound heterozygous mice carrying one fII(lox) allele and one constitutive-null allele were viable. Young adults exhibited neither spontaneous bleeding events nor diminished reproductive success. However, the induction of Cre recombinase in fII(lox) mice using the poly I:C-inducible Mx1-Cre system resulted in the rapid and near-complete recombination of the fII(lox) allele within the liver, the loss of circulating prothrombin, and profound derangements in coagulation function. Consistent with the notion that thrombin regulates coagulation and inflammatory pathways, an additional early consequence of reducing prothrombin was impaired antimicrobial function in mice challenged with Staphylococcus aureus peritonitis. However, life expectancy in unchallenged adults genetically depleted of prothrombin was very short ( approximately 5-7 days). The loss of viability was associated with the development of severe hemorrhagic events within multiple tissues, particularly in the heart and brain. Unlike the constitutive loss of either clotting or platelet function alone, the conditional loss of prothrombin is uniformly not compatible with maintenance of hemostasis or long-term survival.

Pediatric faculty members' attitudes about part-time faculty positions and policies to support part-time faculty: a study at one medical center.

PURPOSE: To examine pediatric faculty members' attitudes about part-time faculty positions and policies to support part-time faculty. METHOD: In 2001, an anonymous 26-item questionnaire assessing attitudes about part-time faculty was mailed to all 441 faculty members of Cincinnati Children's Hospital Medical Center. Multivariable analyses were used to determine faculty characteristics associated with specific attitudes, and qualitative methods were used to analyze responses to an open-ended item assessing beliefs about facilitating part-time careers. RESULTS: Three hundred (68%) faculty members completed questionnaires. Twenty-nine (10%) worked part-time and an additional 88 (33%) had considered part-time work, primarily because of dependent children. Although 177 (59%) believed that part-time faculty were perceived as being less committed to their careers and the institution, 207 (69%) believed part-time faculty should be eligible for all academic tracks and 219 (73%) that they should be allowed extension of time to obtain tenure. Most reported that policy changes to support part-time faculty would enhance diversity (N = 234, 78%) and improve recruitment, retention, and promotion of female faculty. Multivariable analysis demonstrated that women and respondents with dependent children were more likely to be concerned about perceived commitment and more likely to endorse policies to support part-time faculty. Participants suggested that part-time careers for faculty would be facilitated by clarifying productivity expectations, expanding resources, and modifying existing policies. CONCLUSIONS: Although women and respondents with dependent children were concerned about perceived commitment of part-time faculty and were most supportive of policies that would support part-time faculty, pediatric faculty generally supported such policies.

Rescue of prothrombin-deficiency by transgene expression in mice.

Prothrombin has diverse biological functions in addition to its well established role in blood coagulation. In order to study these functions in more detail mouse model systems are needed. Since deficiency of prothrombin in mice results in partial embryonic lethality and neonatal death, alternative approaches are required to study the biology of prothrombin in the adult mouse. The liver is the major site of synthesis of prothrombin and therefore liver-specific promoters were used to express prothrombin in transgenic mice. Mice generated from crosses with these transgenic mice and mice hemizygous for the knock-out allele were used to test whether liver-specific expression is sufficient to correct the phenotype of null mice and whether liver-specific expression is sufficient for the development and survival of mice to adulthood. The mouse albumin promoter/enhancer was used initially for transgene expression without success in obtaining transgene positive, endogenous prothrombin null mice. Two lines of transgene positive, endogenous prothrombin deficient mice were obtained using the mouse transthyretin (TTR) promoter/enhancer driving expression of a human prothrombin cDNA. One line was able to rescue both the embryonic and the neonatal lethality while the other line was only able to correct the embryonic lethality. Expression of prothrombin was restricted to the liver and stomach in one line and to the liver, pancreas, stomach and kidney in the other line of mice. Thrombin activity for one line was determined to be at 5-10% of wildtype levels. These mice developed normally and did not have spontaneous bleeding events unless traumatized. Therefore, transgenic expression of human prothrombin is sufficient for the rescue of the lethality found for prothrombin deficiency in mice.

Cis-acting elements in the hepatocyte growth factor-like protein gene regulate kidney and liver-specific expression in mice.

Previous studies from our laboratory demonstrated that the hepatocyte-specific transcriptional activity of the hepatocyte growth factor-like protein/macrophage stimulating protein (HGFL) promoter is modulated in HepG2 cells by the first 135 base pairs (bp) upstream of the HGFL transcriptional start site. Gel mobility shift and transactivation assays demonstrated that hepatocyte nuclear factor-4 (HNF-4) binds to this region and is responsible, in part, for the liver-specific expression of this gene in HepG2 cells. In an attempt to understand the in vivo mechanism regulating the expression of HGFL, a series of transgenic mice were generated that contained four different regions upstream of the HGFL promoter attached to the coding sequences for chloramphenicol acetyltransferase (CAT). Interestingly, upstream promoter sequences, containing as little as 104 bp upstream of the translational start site, were able to drive reporter expression and protein production specifically in kidney and liver tissue. Strikingly, when the first exon and intron of the HGFL gene was inserted downstream of the 135 bp promoter element, only liver-specific expression was observed. These studies indicate that short sequences upstream of HGFL can drive efficient expression in kidney and liver tissue, and that sequences in the first intron of the HGFL gene contain regulatory elements that direct kidney-specific transcriptional repression in vivo and aid in the proper recapitulation of HGFL expression in mice.

The receptor tyrosine kinase Ron is expressed in the mouse ovary and regulates inducible nitric oxide synthase levels and ovulation.

OBJECTIVE: To determine the reproductive effects in mice of the deletion of the tyrosine kinase domain of the Ron receptor. DESIGN: Controlled animal studies. SETTING: Academic research environment. ANIMAL(S): Immature mice with deletion of the tyrosine kinase domain of the Ron receptor (TK-/-) at 22-30 days of age and adult black Swiss female mice at 5-6 weeks of age. INTERVENTION(S): Hormonal stimulation of immature female TK-/- animals to induce ovulation. MAIN OUTCOME MEASURE(S): Ovulation rates measured by counting the total number of cumulus oocyte complexes in the ampullar region of the murine oviduct after hormonal stimulation. Western blot analysis measured murine ovarian protein levels of endothelial and inducible nitric oxide synthase (iNOS). Immunohistochemical analysis localized iNOS in the developing murine ovarian follicle. RESULT: Immature TK-/- mice (22-30 days) ovulate significantly fewer cumulus oocyte complexes. Western blot analyses demonstrated increased levels of iNOS before and after ovulation compared with controls. Conversely, endothelial nitric oxide synthase levels were similar and remained constant during corresponding time periods. Immunohistochemical analyses demonstrated a significant increase in iNOS staining throughout the ovary in TK-/- mice with a significant amount of iNOS in granulosa cells surrounding the oocyte when compared with controls. CONCLUSION(S): The increased level of nitric oxide in the TK-/- mice is likely due to an elevated level of iNOS, which may contribute to a decrease in the size of the ovaries and ovulation rates of immature TK-/- animals.

Short-form Ron receptor is required for normal IFN-gamma production in concanavalin A-induced acute liver injury.

Abrogation of Ron receptor tyrosine kinase function results in defects in macrophage activation and dysregulated acute inflammatory responses in vivo. Several naturally occurring constitutively active alternative forms of Ron have been identified, including from primary human tumors and tumor cell lines. One of these alternative forms, short-form (SF) Ron, is generated from an alternative start site in intron 10 of the Ron gene that eliminates most of the extracellular portion of the receptor and is overexpressed in several human cancers. To test the physiological significance of SF-Ron in vivo, mice were generated that solely express the full-length form of Ron (FL-Ron). Our results show that elimination of the capacity to express SF-Ron in vivo leads to augmented production of IFN-gamma from splenocytes following stimulation ex vivo with either concanavalin A or anti-CD3/T cell receptor monoclonal antibody. Moreover, in a concanavalin A-induced murine model of acute liver injury, FL-Ron mice have increased production of serum INF-gamma and serum alanine aminotransferase levels and worsened liver histology and overall survival compared with wild-type control mice. Taken together, these results suggest for the first time that SF-Ron impacts the progression of inflammatory immune responses in vivo and further support a role for the Ron receptor and its various forms in liver pathophysiology.

Cross-talk between the receptor tyrosine kinases Ron and epidermal growth factor receptor.

Heterogeneous receptor-receptor interactions may play a role in intracellular signaling. Accordingly, the interaction of two dissimilar tyrosine kinase receptors, Ron and epidermal growth factor receptor (EGFR) was investigated. The functional interaction of Ron and EGFR in cell scatter and oncogenic transformation was investigated in vivo. Transfection of a dominant negative form of EGFR into human embryonic kidney cells stably expressing Ron (293-Ron) dramatically reduced the scatter response induced by the Ron ligand hepatocyte growth factor-like protein/macrophage stimulating protein (HGFL). The scatter response of the 293-Ron cells was also attenuated by treatment of the cells with the specific EGFR inhibitor AG 1478. Co-transfection of Ron and dominant-negative EGFR, or co-transfection of EGFR and a dominant-negative form of Ron reduced focus formation in NIH/3T3 cells. Western analysis of NIH/3T3 cells overexpressing murine Ron and expressing endogenous levels of EGFR was used to demonstrate that Ron and EGFR co-immunoprecipitate. Stimulation of the cells in vitro with the Ron ligand HGFL or with the EGFR ligand epidermal growth factor (EGF) appeared to induce phosphorylation of both receptors. Co-immunoprecipitation and phosphorylation of phosphatidyl inositol 3-kinase (PI3-K) was also observed. This novel finding of a functional and biochemical interaction between Ron and EGFR suggests that heterologous tyrosine kinase receptor interactions may play a role in cellular processes such as scatter and transformation.

Deletion of the Ron receptor tyrosine kinase domain in mice provides protection from endotoxin-induced acute liver failure.

The targeted deletion of the cytoplasmic domain of the Ron receptor tyrosine kinase (TK) in mice leads to exaggerated responses to injury in several murine models of inflammation as well as increased lethality in response to endotoxin (lipopolysaccharide [LPS]). Using a well-characterized model of LPS-induced acute liver failure (ALF) in galactosamine (GalN)-sensitized mice, we show that Ron TK(-/-) mice display marked protection compared with control Ron TK(+/+) mice. Whereas control mice have profound elevation of serum aminotransferase levels (a marker of hepatocyte injury) and hemorrhagic necrosis of the liver, in dramatic contrast, Ron TK(-/-) mice have mild elevation of aminotransferase levels and relatively normal liver histology. These findings are associated with a reduction in the number of liver cells undergoing apoptosis in Ron TK(-/-) mice. Paradoxically, treatment of Ron TK(-/-) mice with LPS/GalN leads to markedly elevated (3.5-fold) serum levels of tumor necrosis factor (TNF) alpha, a key inflammatory mediator in this liver injury model, as well as reduced amounts of interleukin (IL) 10 (a suppressor of TNF-alpha production) and interferon (IFN)-gamma (a TNF-alpha sensitizer). These results show that ablation of the TK activity of the Ron receptor leads to protection from the development of hepatocellular apoptosis in response to treatment with LPS/GalN, even in the presence of excessive levels of serum TNF-alpha. In conclusion, our studies show that the Ron receptor TK plays a critical role in modulating the response of the liver to endotoxin.

Receptor tyrosine kinase Ron is expressed in mouse reproductive tissues during embryo implantation and is important in trophoblast cell function.

Ron is a receptor tyrosine kinase that is activated by the binding of hepatocyte growth factor-like (HGFL) protein. Mutations in the catalytic domain of this receptor result in an aggressively invasive phenotype. Conversely, deletion of the entire receptor results in an embryonic lethality by Embryonic Day 7.5. The specific cellular localization and mechanisms of action of Ron and HGFL during embryo implantation are not known. Therefore, this report characterizes the temporal and spatial distribution of this receptor during mouse embryo implantation and placentation. Reverse transcription-polymerase chain reaction analysis demonstrated the presence of Ron transcripts in the uterus, placenta, testis, and epididymis, whereas HGFL transcripts were found in the cervix, placenta, epididymis, and testis. In situ hybridization and immunohistochemical analyses demonstrated that Ron was present in the cells of the ectoplacental cone and trophoblast giant cell regions surrounding the implanting embryo. Ron expression was also observed in SM9-1, SM9-2, and SM-10 murine trophoblast cell lines. To determine the effects of Ron activation on trophoblast function, Matrigel invasion and cell survival assays were performed using the SM9-1 and SM-10 trophoblast cell lines. The HGFL stimulation of these cells increased invasion and enhanced cell survival. These observations suggest that activation of the Ron receptor by HGFL binding may aid in implantation by way of trophoblast function and viability.

Assessment of recombinant porcine follicle-stimulating hormone receptor using a novel polyclonal ectodomain antibody.

Follicle-stimulating hormone (FSH) receptors (FSHR) are critically involved in mediating the responses of granulosa cells and Sertoli cells to FSH. The dynamic changes in cell surface FSH receptors (FSHR) in response to FSH remain unclear in part because of the heavy reliance on ligand-binding methodologies. This study was designed to determine the molecular and cellular properties of recombinant porcine FSHR using a novel, high-affinity purified polyclonal antibody to the ectodomain of the pFSHR. A full-length porcine FSHR cDNA was cloned and sequenced and recombinant pFSHR protein was stably expressed in a clonal cell line of Chinese hamster ovary cells (pFSHR-CHO). Recombinant receptor was stably expressed in an ovarian cell line with a density similar to that of porcine ovarian cells. A specific polyclonal antibody was generated in chickens to a 100-amino acid fragment of the pFSHR ectodomain. Immunoblotting, immunoprecipitation, indirect immunofluorescence cytochemistry and immunoelectron microscopy were performed using affinity-purified antibody to identify recombinant pFSHR in pFSHR-CHO cells. Immunoblotting of solubilized pFSHR-CHO proteins and immunoprecipitation of pFSHR-CHO protein metabolically labeled with 35S identified a single 74-kDa band in pFSHR-CHO cells; no bands were visualized in mock-transfected CHO cells. Indirect immunofluorescent labeling revealed the presence of pFSHR in pFSHR-CHO cells but not in mock-transfected CHO cells. Immunoelectron microscopy revealed the highest density of pFSHR associated with the plasma membrane and no pFSHR in mock-transfected CHO cells. The chicken anti-pFSHR antibody is a valuable tool for detecting and monitoring of FSHR using a variety of methodologies.

The role of the receptor tyrosine kinase Ron in nickel-induced acute lung injury.

Acute lung injury (ALI), a severe respiratory syndrome, develops in response to numerous insults and responds poorly to therapeutic intervention. Recently, cDNA microarray analyses were performed that indicated several pathogenic responses during nickel-induced ALI, including marked macrophage activation. Macrophage activation is mediated, in part, via the receptor tyrosine kinase Ron. To address the role of Ron in ALI, the response of mice deficient in the cytoplasmic domain of Ron (Ron tk-/-) were assessed in response to nickel exposure. Ron tk-/- mice succumb to nickel-induced ALI earlier, express larger, early increases in interleukin-6, monocyte chemoattractant protein-1, and macrophage inflammatory protein-2, display greater serum nitrite levels, and exhibit earlier onset of pulmonary pathology and augmented pulmonary tyrosine nitrosylation. Increases in cytokine expression and cellular nitration can lead to tissue damage and are consistent with the differences between genotypes in the early onset of pathology and mortality in Ron tk-/- mice. These analyses indicate a role for the tyrosine kinase receptor Ron in ALI.