Point mutations and overexpression of Ron induce transformation, tumor formation, and metastasis.

The receptor tyrosine kinase Ron is a member of the receptor family that includes the proto-oncogene Met and the avian oncogene Sea. The interaction of Ron with its ligand, known as hepatocyte growth factor-like protein (HGFL) or macrophage stimulating protein (MSP), induces crucial cellular responses including invasive growth, proliferation, cell scattering, and branching morphogenesis. Based on the homology and functional similarities between Met and Ron it was hypothesized that Ron may be important in tumor formation and metastasis. To test this hypothesis, wild-type mouse Ron and three mutant forms of Ron containing mutations similar to those found in the Met gene in human hereditary papillary renal carcinoma (HPRC), were expressed in NIH3T3 cells. A transformed phenotype was produced in cell lines expressing either wild-type Ron or the mutated Ron proteins. Further, these cell lines displayed oncogenic potential by exhibiting increased proliferation and constitutive phosphorylation of Ron. These cell lines were also tested for the ability to form solid tumors. Cells expressing wild-type Ron and the three proteins with single amino acid substitutions were highly tumorigenic in vivo. In a model of experimental metastasis, two of the cell lines with altered Ron protein formed highly aggressive tumors in the lungs. These results suggest that Ron may be an aggressive oncogene when either overexpressed or when activated by mutation.

Ron-mediated cytoplasmic signaling is dispensable for viability but is required to limit inflammatory responses.

Ron receptor activation induces numerous cellular responses in vitro, including proliferation, dissociation, and migration. Ron is thought to be involved in blood cell development in vivo, as well as in many aspects of the immune response including macrophage activation, antigen presentation, and nitric oxide regulation. In previous studies to determine the function of Ron in vivo, mice were generated with a targeted deletion of the extracellular and transmembrane regions of this gene. Mice homologous for this deletion appear to die early during embryonic development. To ascertain the in vivo function of Ron in more detail, we have generated mice with a germline ablation of the tyrosine kinase domain. Strikingly, our studies indicate that this domain of Ron, and therefore Ron cytoplasmic signaling, is not essential for embryonic development. While mice deficient in this domain are overtly normal, mice lacking Ron signaling have an altered ability to regulate nitric oxide levels and, in addition, have enhanced tissue damage following acute and cell-mediated inflammatory responses.

Prothrombin Scranton: substitution of an amino acid residue involved in the binding of Na+ (LYS-556 to THR) leads to dysprothrombinemia.

Several members of a family from Scranton, Pennsylvania were identified to have normal levels of prothrombin antigen while their prothrombin clotting activity was approximately 50% of normal. There has been no previous history of bleeding or other clinical manifestations in this family. The genomic DNA from the proband was amplified for all exons in the prothrombin gene and analyzed by single strand conformation polymorphism (SSCP)/heteroduplex analysis followed by DNA sequence analysis and restriction enzyme digestion. A mutation at nucleotide 20040 in exon 14 was identified and confirmed by restriction enzyme digestion. This mutation results in the substitution of Thr for Lys at amino acid 556. Amino acid 556 has been reported as one of the key residues for the binding of Na+ in the thrombin portion of the protein.

Gene targeting in hemostasis. Prothrombin.

There have been extensive studies on the structure and function of prothrombin; a protein critical for the coagulation of blood. The biological functions of prothrombin and its activated form, thrombin are discussed, as well as the structure and functional domains of the protein. Prothrombin is expressed in a tissue-specific manner and its gene structure and regulatory elements have been analyzed in detail. In order to learn more about the functions of prothrombin in an in vivo context, the gene was ablated in mice. Homozygous deletion of prothrombin results in a partial embryonic lethal phenotype. Approximately half of the homozygous mutant mice die during mid-gestation and the remainder die soon after birth. The cause of death of neonates is due to excessive bleeding, while null embryos have a lack of integrity of the yolk sac membrane resulting in bleeding into the yolk sac cavity. These results are discussed in relation to the phenotypes found for other mice lacking specific coagulation factors.

Prothrombin San Antonio: a single amino acid substitution at a factor Xa activation site (Arg320 to His) results in dysprothrombinemia.

Three members of a San Antonio, Texas, family were identified with prothrombin activity levels half the normal level but to have normal levels of antigen. All exons of the prothrombin gene from the proband were sequenced. A G-to-A mutation at nucleotide 7543 was found that resulted in the substitution of His for Arg at residue 320. The Arg320-Ile321 bond is 1 of 2 sites in prothrombin cleaved by Factor Xa in the prothrombinase complex to form thrombin. Substitution of His for Arg at this site resulted in the blockage of Factor Xa cleavage, forming a dysfunctional molecule. The proband, her mother, and her maternal aunt were found to be heterozygous for this mutation. This is the first known observation of an amino acid substitution at this site that resulted in dysprothrombinemia. (Blood. 2000;95:711-714)

Prothrombin carora: hypoprothrombinaemia caused by substitution of Tyr-44 by Cys.

Two members of a family from Carora, Venezuela, were found to have prothrombin activity levels at 4% of normal and undetectable antigen levels. All exons of the prothrombin gene from the proband were sequenced and a mutation at nucleotide 1305 was identified that would result in the substitution of Cys for Tyr at residue 44. Residue 44 is present in the aromatic stack region of the protein. Substitution of a Cys in this region would result in an abnormal folding of the protein which could be the cause for the observed lack of secretion of the abnormal prothrombin.

The Ron/STK receptor tyrosine kinase is essential for peri-implantation development in the mouse.

The Ron/STK receptor tyrosine kinase is a member of the c-Met family of receptors and is activated by hepatocyte growth factor-like protein (HGFL). Ron activation results in a variety of cellular responses in vitro, such as activation of macrophages, proliferation, migration, and invasion, suggesting a broad biologic role in vivo. Nevertheless, HGFL-deficient mice grow to adulthood with few appreciable phenotypic abnormalities. We report here that in striking contrast to the loss of its only known ligand, complete loss of Ron leads to early embryonic death. Embryos that are devoid of Ron (Ron-/-) are viable through the blastocyst stage of development but fail to survive past the peri-implantation period. In situ hybridization analysis demonstrates that Ron is expressed in the trophectoderm at embryonic day (E) 3.5 and is maintained in extraembryonic tissue through E7.5, compatible with an essential function at this stage of development. Hemizygous mice (Ron+/-) grow to adulthood; however, these mice are highly susceptible to endotoxic shock and appear to be compromised in their ability to downregulate nitric oxide production. These results demonstrate a novel role for Ron in early mouse development and suggest that Ron plays a limiting role in the inflammatory response.

Synthesis of dihydroxanthone derivatives and evaluation of their inhibitory activity against acetylcholinesterase: unique structural analogs of tacrine based on the BCD-ring of arisugacin.

A general approach to synthesis of dihydroxanthone derivatives is described here. In vitro evaluation of these dihydroxanthones demonstrated that some derivatives possess moderate anti-cholinesterase activities and better selectivities than tacrine for acetylcholinesterase over butyrylcholinesterase. Structural effects on anti-cholinesterase activities were also examined, and docking experiments were carried out to provide preliminary understandings of these experimental observations.

Expression of hepatocyte growth factor-like protein is repressed by retinoic acid and enhanced by cyclic adenosine 3',5'-monophosphate response element-binding protein (CREB)-binding protein (CBP).

In an effort to understand the molecular mechanisms involved in the regulation of expression of the gene encoding hepatocyte growth factor-like protein (HGFL), it was found that all-trans-retinoic acid dramatically represses expression of the endogenous HGFL gene in HepG2 cells, a human hepatocyte-derived cell line. This repression requires the sequence between nucleotides -135 and -105 in the 5'-flanking sequence of the HGFL gene, a site that has previously been shown to bind the transcription factor hepatocyte nuclear factor-4 (HNF-4). Electrophoretic mobility shift analysis suggests that the retinoic acid receptor does not bind to this site, and that retinoic acid does not alter binding of HNF-4 to this DNA site. However, the transcriptional coactivator, CREB-binding protein (CBP) coactivates expression of this gene through an indirect interaction with the HNF-4-binding site, and overexpression of CBP in HepG2 cells eliminates retinoic acid repression of reporter gene expression driven by the HGFL promoter. Overexpression of CBP also protects the endogenous HGFL gene from down-regulation by retinoic acid. These results suggest that HGFL gene expression requires CBP, and competition for limiting amounts of CBP by retinoic acid receptor may be a means of modifying the activity of HNF-4 at the HGFL gene promoter.

The biology of prothrombin.

Prothrombin and thrombin are involved in diverse biological functions. The structure of prothrombin has been studied extensively and its cDNA has been cloned from several species. The tissue-specific expression of this protein has been studied, as well as the developmental expression pattern. The structure of the human gene coding for prothrombin has been determined, and gene regulation studies have been performed that indicate that HNF-1 might be responsible for the liver-specific expression of this protein. Other regulatory elements have been identified. In order to further study the biological properties of prothrombin, prothrombin-deficient mice have been generated using gene targeting technology. Prothrombin deficiency in mice results in partial embryonic lethality. The mice that survive to birth die from bleeding events. The embryonic lethality occurs between embryonic days 9.5 and 11.5 and appears to be due to the loss of integrity of the vasculature due to a failure in blood coagulation. These results indicate that prothrombin plays not only a key role in hemostasis but suggests that it may be important for mouse development.

Prothrombin deficiency results in embryonic and neonatal lethality in mice.

The conversion of prothrombin (FII) to the serine protease, thrombin (FIIa), is a key step in the coagulation cascade because FIIa triggers platelet activation, converts fibrinogen to fibrin, and activates regulatory pathways that both promote and ultimately suppress coagulation. However, several observations suggest that FII may serve a broader physiological role than simply stemming blood loss, including the identification of multiple G protein-coupled, thrombin-activated receptors, and the well-documented mitogenic activity of FIIa in in vitro test systems. To explore in greater detail the physiological roles of FII in vivo, FII-deficient (FII-/-) mice were generated. Inactivation of the FII gene leads to partial embryonic lethality with more than one-half of the FII-/- embryos dying between embryonic days 9.5 and 11.5. Bleeding into the yolk sac cavity and varying degrees of tissue necrosis were observed in many FII-/- embryos within this gestational time frame. However, at least one-quarter of the FII-/- mice survived to term, but ultimately they, too, developed fatal hemorrhagic events and died within a few days of birth. This study directly demonstrates that FII is important in maintaining vascular integrity during development as well as postnatal life.

Biological effects of targeted inactivation of hepatocyte growth factor-like protein in mice.

Hepatocyte growth factor-like protein (HGFL) is a liver-derived serum glycoprotein involved in cell proliferation and differentiation, and is proposed to have a fundamental role in embryogenesis, fertility, hematopoiesis, macrophage activation, and tissue repair. To assess the in vivo effects of total loss of HGFL, we generated mice with targeted disruption of the gene resulting in loss of the protein. Disruption of the HGFL gene allowed for normal embryogenesis, and followed a Mendelian pattern of genetic transmission. Mice homozygous for the targeted allele (HGFL-/- mice) are fertile, and grow to adulthood without obvious phenotypic abnormalities in unchallenged animals, except for development of lipid-containing cytoplasmic vacuoles in hepatocytes throughout the liver lobules. These histologic changes are not accompanied by discernible changes in synthetic or excretory hepatic functions. Hematopoiesis appears unaltered, and although macrophage activation is delayed in the absence of HGFL, migration to the peritoneal cavity upon challenge with thioglycollate was similar in HGFL-/- and wild-type mice. Challenged with incision to skin, HGFL-/- mice display normal wound healing. These data demonstrate that HGFL is not essential for embryogenesis, fertility, or wound healing. HGFL-deficient mice will provide a valuable means to assess the role of HGFL in hepatic and systemic responses to inflammatory and infectious stimuli in vivo.

Characterization of the mouse Ron/Stk receptor tyrosine kinase gene.

In an effort to understand the mechanisms governing the regulation of the mouse Ron receptor gene, a mouse genomic library was screened and overlapping clones coding for the Ron gene and flanking DNA were identified. Continuous DNA sequence was obtained for approximately 16.4 kilobases. The gene, from the initiator methionine to the polyadenylation site, is contained within 13 244 basepairs and contains 19 exons. Primer extension analyses were performed to determine the transcription start site of the mouse Ron transcript. Multiple transcription start sites were found which also appear to be used in transfected reporter constructs containing Ron 5' flanking DNA. To determine the location of sites which may be critical for the function of the Ron gene promoter, a series of chimeric genes containing serial deletions of the Ron gene promoter fused to the coding sequences for the chloramphenicol acetyl-transferase gene were constructed. Transient transfection analyses of these hybrid genes into various cell lines demonstrated that two regions of the Ron gene promoter, encompassing nucleotides -585 to -465 and from -465 to -285, are important for expression of this transcript in CMT-93 cells. Further analysis of the Ron promoter utilizing gel mobility shift analyses suggests that regions encompassing nucleotides -585 to - 508 and nucleotides -375 to -285 appear to bind specific proteins which may be involved in the negative and positive regulation, respectively, of the mouse Ron gene.

Structure of the human D1F15S1A locus: a chromosome 1 locus with 97% identity to the chromosome 3 gene coding for hepatocyte growth factor-like protein.

The human chromosome 3 locus coding for hepatocyte growth factor-like protein/macrophage stimulating protein (HGFL/MSP) is homologous to two sets of amplified loci on human chromosome 1 at 1p36. One copy of one of the amplified loci (D1F15S1A) has been further characterized by restriction enzyme and DNA sequence analysis. A total of 8331 bp of continuous sequence was determined for this locus. The first 6878 bp of sequence is 96.1% identical to the HGFL/MSP gene, while there is no homology between the two genes following nucleotide 6878. Based on the presence of a 5 bp deletion in putative exon 2 and several downstream stop codons it is very likely that this gene is a pseudogene. Screening of a human liver cDNA library with a chromosome 1-specific probe indicates that at least several other members of the chromosome 1 loci are transcribed.

Functional characterization of domains contained in hepatocyte growth factor-like protein.

To delineate the functional protein domains necessary for the biological activity of hepatocyte growth factor-like protein (HGFL), we created various site-directed and deletion mutated cDNAs coding for this protein. Wild-type and mutated versions of HGFL were produced after transfection of the corresponding cDNAs into tissue culture cells. The biological importance of the domains within HGFL was then examined by addition of recombinant wild-type or mutant forms of HGFL to assays aimed at elucidating regions involved in the stimulation of DNA synthesis, the induction of shape changes in macrophages, and the ability to stimulate cell scattering. Mutant proteins lacking the serine protease-like domain (light chain) were not biologically active in any of the assays tested and could not compete with wild-type HGFL in cell scattering experiments. These data, in addition to direct enzyme-linked immunosorbent assay analyses, suggest that the light chain may play an important role in the interaction of HGFL with its receptor, Ron. Elimination of the proposed protease cleavage site between the heavy and light chains (by mutation of Arg-483 to Glu) produced a protein with activity comparable to wild-type HGFL. Further studies with this mutated protein uncovered an additional proteolytic cleavage site that produces biologically active protein. Deletion of the various kringle domains or the amino-terminal hairpin loop had various effects in the multiple assays. These data suggest that the heavy chain may play a pivotal role in determining the functional aspects of HGFL.

Hepatocyte nuclear factor-4 is responsible for the liver-specific expression of the gene coding for hepatocyte growth factor-like protein.

In an attempt to understand the molecular mechanism regulating the expression of the gene coding for human hepatocyte growth factor-like protein/macrophage stimulating protein (HGFL), our laboratory has isolated and characterized approximately 4200 bp of the 5'-flanking region of the HGFL gene. To determine the location of sites which may be critical for the function of the HGFL gene promoter, we constructed a series of hybrid genes containing serial deletions of this region attached to the coding sequences for chloramphenicol acetyltransferase. Expression of these chimeric plasmids was examined by transient transfection of HepG2 and 293 cells. Our results suggest that the transcriptional activity of the HGFL promoter is modulated in HepG2 cells by one positive element at position -135 to -105 (-135/-105). In contrast, only background levels of chloramphenicol acetyltransferase expression have been detected in 293 cells. The -135/-105 region appears to bind a liver-specific transcription factor essential for expression of this gene. Gel mobility shift experiments with antibodies against hepatocyte nuclear factor-4 (HNF-4) and transactivation of the HGFL promoter by a HNF-4 cDNA expression vector suggest that HNF-4 binds to the -135/-105 region and is responsible for the liver-specific expression of HGFL.

Developmental expression of protein C and protein S in the rat.

In order to better understand the expression of the Protein C/Protein S anticoagulant system, we have isolated and characterized cDNAs coding for rat Protein C and Protein S. These cDNAs were used in Northern analysis to determine tissue-specificity and developmental expression patterns for mRNAs coding for Proteins C and S. In rats, Protein C mRNA is expressed almost exclusively in liver with a small amount of expression in kidney, diaphragm, stomach, intestine, uterus and placenta. Protein C mRNA was not expressed in brain, heart, lung, spleen, small intestine, large intestine, ovary, or urinary bladder. In liver, Protein C mRNA is expressed at very low levels at prenatal day 18 and these levels increased to maximal levels by postnatal day 13. The size of the mRNA coding for rat Protein C is approximately 1.9 kb. Rat Protein S mRNA was expressed in all tissues examined: brain, heart, lung, diaphragm, liver, spleen, stomach, small intestine, large intestine, kidney, adrenal ovary, uterus, placenta, and urinary bladder. Interestingly, there were 4 bands hybridizing with the rat protein S cDNA that were evident in many of the tissues examined, corresponding to mRNA sizes of approximately 3.5, 2.6, 1.8, and 0.3 kb. There was a difference in tissue-specificity of each mRNA. The 1.8 kb band is generally the most prominent autoradiographic band in any tissue. From these results, it is evident that the expression of Protein C mRNA is similar to that of other vitamin K-dependent proteins. The expression of Protein S mRNA, however, is surprisingly complex and may include alternative splicing of mRNA to generate the various sizes evident on Northern analysis.

Prothrombin Frankfurt: a dysfunctional prothrombin characterized by substitution of Glu-466 by Ala.

We have identified a patient with a dysfunctional prothrombin that we have designated Prothrombin Frankfurt. The proband was characterized by a prothrombin activity level of 13% and 20% compared to normal controls using two different assays with a normal prothrombin antigen level of 91% of normal controls. The genetic defect responsible for the abnormal prothrombin activity was determined by the polymerase chain reaction followed by single-strand conformation polymorphism (PCR-SSCP) analysis and by DNA sequence analysis of the human prothrombin gene. Substitution of a C for an A at nucleotide 10177 in the human prothrombin gene of the proband was identified, which results in the replacement of Glu-466 by Ala. The proband and one sister were homozygous for this mutation. Both parents, as well as one brother, were found to be heterozygous for this mutation. The same amino acid substitution was previously identified to be responsible for the dysfunctional protein Prothrombin Salakta and was hypothesized to result in altered substrate specificity. Four polymorphisms were also identified in the prothrombin gene from the proband when compared to the published sequence at nucleotides 554, 4048, 4272 and 10253.

Increased expression of mRNA for hepatocyte growth factor-like protein during liver regeneration and inflammation.

To assess potential roles of hepatocyte growth factor-like (HGFL) protein as a growth factor and as a cytokine, we determined the expression of mRNA for (HGFL) protein during liver regeneration and inflammation. Expression of mRNA for HGFL protein in the regenerating liver was upregulated to 93% and 64% above controls at 1 h following partial hepatectomy and carbon tetrachloride treatment of rats, respectively. Similarly, rat liver mRNA for HGFL protein was upregulated to 78% above controls at 24 h after injection of thioglycollate (inducing activation of peritoneal macrophages) and to 118% at 48 h after injection of turpentine (inducing the acute phase response). These results support a potential role for HGFL protein in the early phase of liver regeneration and as an inflammatory mediator.

Hepatic expression of hepatocyte-growth-factor-like/macrophage-stimulating protein mRNA in fulminant hepatic failure.

We investigated whether impaired Kupffer cell phagocytosis in fulminant hepatic failure could be due to reduced synthesis of hepatocyte-growth-factor-like/macrophage-stimulating protein (HGFL/MSP), a serum protein synthesised predominantly in hepatocytes and required by tissue macrophages for phagocytosis. Hepatic expression of the 3.0 kb HGFL/MSP mRNA, assessed by northern hybridisation, was lower in nine patients with fulminant hepatic failure undergoing liver transplantation than in three liver grafts as controls (median absorbance units 97 [range 15-1200] versus 1114 [1100-1120], respectively; p < 0.05). Decreased hepatic HGFL/MSP production might cause impaired Kupffer cell phagocytosis in fulminant hepatic failure.