Biomedical subjects

Wallace L McKeehan

Publications and source records attributed to Wallace L McKeehan.

At least 19 recordsLinked to original sources

PubMed2007

Fibroblast growth factor receptor 2 tyrosine kinase is required for prostatic morphogenesis and the acquisition of strict androgen dependency for adult tissue homeostasis.

The fibroblast growth factor (FGF) family consists of 22 members and regulates a broad spectrum of biological activities by activating diverse isotypes of FGF receptor tyrosine kinases (FGFRs). Among the FGFs, FGF7 and FGF10 have been implicated in the regulation of prostate development and prostate tissue homeostasis by signaling through the FGFR2 isoform. Using conditional gene ablation with the Cre-LoxP system in mice, we demonstrate a tissue-specific requirement for FGFR2 in urogenital epithelial cells--the precursors of prostatic epithelial cells--for prostatic branching morphogenesis and prostatic growth. Most Fgfr2 conditional null (Fgfr2(cn)) embryos developed only two dorsal prostatic (dp) and two lateral prostatic (lp) lobes. This contrasts to wild-type prostate, which has two anterior prostatic (ap), two dp, two lp and two ventral prostatic (vp) lobes. Unlike wild-type prostates, which are composed of well developed epithelial ductal networks, the Fgfr2(cn) prostates, despite retaining a compartmented tissue structure, exhibited a primitive epithelial architecture. Moreover, although Fgfr2(cn) prostates continued to produce secretory proteins in an androgen-dependent manner, they responded poorly to androgen with respect to tissue homeostasis. The results demonstrate that FGFR2 is important for prostate organogenesis and for the prostate to develop into a strictly androgen-dependent organ with respect to tissue homeostasis but not to the secretory function, implying that androgens may regulate tissue homeostasis and tissue function differently. Therefore, Fgfr2(cn) prostates provide a useful animal model for scrutinizing molecular mechanisms by which androgens regulate prostate growth, homeostasis and function, and may yield clues as to how advanced-tumor prostate cells escape strict androgen regulations.

Androgens↗

PubMed2006

Control of fibroblast growth factor (FGF) 7- and FGF1-induced mitogenesis and downstream signaling by distinct heparin octasaccharide motifs.

Variation in length, disaccharide composition, and sulfation of heparan sulfate (HS) affects fibroblast growth factor (FGF) signaling. However, it is unclear whether the specific distribution of groups within oligosaccharides or random variations in charge density underlies the effects. Recently we showed that a mixture of undersulfated octasaccharides exhibiting 7 and 8 sulfates (7,8-S-OctaF7) generated from heparin had the highest affinity for FGF7 monitored by salt resistance (>0.60 M salt) of octasaccharide-FGF7 complexes. 7,8-S-OctaF7 also had the highest specific activity for formation of a complex with dimeric FGFR2IIIb competent to bind FGF7. Here we show that when endogenous HS was inhibited by chlorate treatment, 7,8-S-OctaF7 specifically supported FGF7-stimulated DNA synthesis and downstream signaling in FGFR2IIIb-expressing mouse keratinocytes. It failed to support FGF1 signaling in both HS-deficient mouse keratinocytes and 3T3 fibroblasts. In contrast, abundant, more highly sulfated and heterogenous mixtures of octasaccharides with lower affinity (0.30-0.60 M salt) for FGF7 supported FGF1-induced signaling in both cell types. In contrast to the two-component 7,8-S-OctaF7 mixture from FGF7, the high affinity octasaccharide fraction from FGF1 was a heterogeneous mixture with components ranging from 8 to 12 sulfates with 11-S-octasaccharides the most abundant. The high affinity fraction exhibited similar properties to the lower affinity fractions from both FGF1 and FGF7. Octasaccharide mixtures eluting from FGF1 between 0.30 and 0.60 M and above 0.60 M salt were nearly equal in support of FGF1 signaling in fibroblasts and keratinocytes. Both were deficient in support of FGF7-induced signaling in keratinocytes. The results show that both variations in overall charge density and specific distribution of charged groups within HS motifs exhibit FGF-specific control over formation of FGF-HS-FGFR complexes and downstream signaling.

3T3 Cells↗

PubMed2006

Structural specificity in a FGF7-affinity purified heparin octasaccharide required for formation of a complex with FGF7 and FGFR2IIIb.

Variations in sulfation of heparan sulfate (HS) affect interaction with FGF, FGFR, and FGF-HS-FGFR signaling complexes. Whether structurally distinct HS motifs are at play is unclear. Here we used stabilized recombinant FGF7 as a bioaffinity matrix to purify size-defined heparin oligosaccharides. We show that only 0.2%-4% of 6 to 14 unit oligosaccharides, respectively, have high affinity for FGF7 based on resistance to salt above 0.6M NaCl. The high affinity fractions exhibit highest specific activity for interaction with FGFR2IIIb and formation of complexes of FGF7-HS-FGFR2IIIb. The majority fractions with moderate (0.30-0.6M NaCl), low (0.14-0.30M NaCl) or no affinity at 0.14M NaCl for FGF7 supported no complex formation. The high affinity octasaccharide mixture exhibited predominantly 7- and 8-sulfated components (7,8-S-OctaF7) and formed FGF7-HS-FGFR2IIIb complexes with highest specific activity. Deduced disaccharide analysis indicated that 7,8-S-OctaF7 comprised of DeltaHexA2SGlcN6S in a 2:1 ratio to a trisulfated and a variable unsulfated or monosulfated disaccharide. The inactive octasaccharides with moderate affinity for FGF7 were much more heterogenous and highly sulfated with major components containing 11 or 12 sulfates comprised of predominantly trisulfated disaccharides. This suggests that a rare undersulfated motif in which sulfate groups are specifically distributed has highest affinity for FGF7. The same motif also exhibits structural requirements for high affinity binding to dimers of FGFR2IIIb prior to binding FGF7 to form FGF7-HS-FGFR2IIIb complexes. In contrast, the majority of more highly sulfated HS motifs likely play FGFR-independent roles in stability and control of access of FGF7 to FGFR2IIIb in the tissue matrix.

Animals↗

PubMed2006

Identification of RNA-binding proteins that regulate FGFR2 splicing through the use of sensitive and specific dual color fluorescence minigene assays.

We have developed a series of fluorescent splicing reporter minigenes for the establishment of cell-based screens to identify splicing regulatory proteins. A key technical advance in the application of these reporters was the use of two different fluorescent proteins: EGFP and monomeric Red Fluorescent Protein (mRFP). Through establishment of stable cell lines expressing such dual color fluorescent reporters, these minigenes can be used to perform enhanced screens for splicing regulatory proteins. As an example of such applications we generated fluorescent minigenes that can be used to determine the splicing of mutually exclusive FGFR2 exons IIIb and IIIc by flow cytometry. One minigene contained a coding sequence for EGFP whose translation was dependent on splicing of exon IIIb, whereas a second minigene required exon IIIc splicing for translation of an mRFP coding sequence. Stable incorporation of both minigenes into cells that express endogenous FGFR2-IIIb or FGFR2-IIIc resulted in EGFP or mRFP fluorescence, respectively. Cells stably transfected with both minigenes were used to screen a panel of cDNAs encoding known splicing regulatory proteins, and several were identified that induced a switch in splicing that could be detected specifically by an increase in green, but not red, fluorescence. We further demonstrated additional minigenes that can be used in dual color fluorescent screens for identification of splicing regulatory proteins that function through specific intronic splicing enhancer elements (ISEs). The methods and minigene designs described here should be adaptable for broader applications in identification of factors and mechanisms involved in alternative splicing of numerous other gene transcripts.

Alternative Splicing↗

PubMed2006

Ectopic activity of fibroblast growth factor receptor 1 in hepatocytes accelerates hepatocarcinogenesis by driving proliferation and vascular endothelial growth factor-induced angiogenesis.

Fibroblast growth factor (FGF) signaling mediates cell-to-cell communication in development and organ homeostasis in adults. Of the four FGF receptor (FGFR) tyrosine kinases, only FGFR4 is expressed in mature hepatocytes. Although FGFR1 is expressed by hepatic cell progenitors and adult nonparenchymal cells, ectopic expression is commonly observed in hepatoma cells. Here, we determined whether ectopic FGFR1 is a cause or consequence of hepatocellular carcinoma by targeting a constitutively active human FGFR1 to mouse hepatocytes. Livers of transgenic mice exhibited accelerated regeneration after partial hepatectomy but no signs of neoplastic or preneoplastic abnormalities for up to 18 months. However, in diethylnitrosamine-treated mice, the chronic FGFR1 activity promoted an incidence of 44% adenomas at 4 months and 38% hepatocellular carcinoma at 8 months. No adenoma or hepatocellular carcinoma was observed in diethylnitrosamine-treated wild-type (WT) livers at 4 or 8 months, respectively. At 10 and 12 months, tumor-bearing livers in transgenic mice were twice the size of those in WT animals. Isolated hepatoma cells from the transgenic tumors exhibited a growth advantage in culture. Advanced hepatocellular carcinoma in the transgenic livers exhibited a reduced rate of necrosis. This was accompanied by a mean microvessel density of 2.7 times that of WT tumors and a markedly higher level of vascular endothelial growth factor. In cooperation with an initiator, the persistent activity of ectopic FGFR1 in hepatocytes is a strong promoter of hepatocellular carcinoma by driving cell proliferation at early stages and promoting neoangiogenesis at late stages of progression.

Animals↗

PubMed2006

Forced expression of hepatocyte-specific fibroblast growth factor 21 delays initiation of chemically induced hepatocarcinogenesis.

Inappropriate fibroblast growth factor (FGF) signaling is involved in most tissue-specific pathologies including cancer. Previously we showed that inappropriate expression and chronic activity of FGF receptor (FGFR) 1 in hepatocytes accelerated diethylnitrosamine (DEN)-initiated hepatocarcinogenesis. Here we showed that although widely expressed FGF1 and FGF2 are frequently upregulated in hepatocellular carcinoma (HCC), germline deletion of both FGF1 and FGF2 had no effect on DEN-initiated hepatocarcinogenesis. Thus overexpression of FGF1 or FGF2 may be a consequence rather than contributor to hepatoma progression. FGF21 is the first of 22 homologues whose expression has been reported to be preferentially in the liver. We showed that similar to FGF1 and FGF2, FGF21 mRNA was upregulated in neoplastic and regenerating liver after partial hepatectomy (PH) and CCl4 administration. In situ hybridization analysis confirmed that in contrast to FGF1 and FGF2, expression of FGF21 mRNA was limited to hepatocytes. Forced overexpression of FGF21 in hepatocytes by gene targeting had no apparent impact on normal liver development and compensatory response to injury. Surprisingly, overexpression of FGF21 delayed the appearance of DEN-induced liver tumors. At 8 and 10 mo, only 10% and 30% of transgenic mice, respectively, developed adenomas compared to 50% (all adenomas) and 80% (60% adenoma/20% HCC) in the wild-type (WT) mice. However, the incidence and burden of HCC at 10 mo and later was equal in the FGF21 transgenic and WT mice. We propose that FGF21 may delay development of adenomas through activation of resident hepatocyte FGFR4 at early times, but counteracts the delay by acceleration of progression to HCC through interaction with ectopic FGFR1 once it appears in hepatoma cells. This indicates a dual function of FGF21 that may reflect changes in FGFR isotype during progression of differentiated hepatoma cells.

Animals↗

PubMed2005

Bile acids decrease hepatic paraoxonase 1 expression and plasma high-density lipoprotein levels via FXR-mediated signaling of FGFR4.

OBJECTIVE: The purpose of this research was to determine how dietary bile acids repress hepatic expression of paraoxonase 1 (PON1). METHODS AND RESULTS: C57BL/6 mice and C3H/HeJ mice, having different susceptibilities to atherosclerosis, were fed a chow diet and an atherogenic diet containing taurocholate. Compared with the more atherosclerosis-susceptible C57BL/6 mice, C3H/HeJ mice display resistance to dietary bile acid repression of hepatic PON1 mRNA and decreased high-density lipoprotein cholesterol. Whereas knockout of toll receptor 4 did not affect response to taurocholate, deletion of either FXR or FGFR4 blocked taurocholate repression of PON1 and CYP7A1. FGF19, an activator of FGFR4 expressed in human ileum, decreased expression of both PON1 and CYP7A1 expression by human hepatoma cells. In all of the mice studied, dietary taurocholate increased ileal expression of FGF15, a FXR-inducible murine homologue of human FGF19. CONCLUSIONS: Hepatic PON1 and CYP7A1 mRNA expression is repressed by bile acids via FXR-mediated induction of FGF15. Thus, the inability of C3H/HeJ mice to display taurocholate repression of PON1 and CYP7A1 mRNAs was not because of a lack of induction of FGF15 but rather signaling events distal to FGF15-FGFR4 association.

Animals↗

PubMed2005

Fgfr4 is required for effective muscle regeneration in vivo. Delineation of a MyoD-Tead2-Fgfr4 transcriptional pathway.

Fgfr4 has been shown to be important for appropriate muscle development in chick limb buds; however, Fgfr4 null mice show no phenotype. Here, we show that staged induction of muscle regeneration in Fgfr4 null mice becomes highly abnormal at the time point when Fgfr4 is normally expressed. By 7 days of regeneration, differentiation of myotubes became poorly coordinated and delayed by both histology and embryonic myosin heavy chain staining. By 14 days much of the muscle was replaced by fat and calcifications. To begin to dissect the molecular pathways involving Fgfr4, we queried the promoter sequences for transcriptional factor binding sites and tested candidate regulators in a 27-time point regeneration series. The Fgfr4 promoter region contained a Tead protein binding site (M-CAT 5'-CATTCCT-3'), and Tead2 showed induction during regeneration commensurate with Fgfr4 regulation. Co-transfection of Tead2 and Fgfr4 promoter reporter constructs into C2C12 myotubes showed Tead2 to activate Fgfr4, and mutation of the M-CAT motif in the Fgfr4 promoter abolished these effects. Immunostaining for Tead2 showed timed expression in myotube nuclei consistent with the mRNA data. Query of the expression timing and genomic sequences of Tead2 suggested direct regulation by MyoD, and consistent with this, MyoD directly bound to two strong E-boxes in the first intron of Tead2 by chromatin immunoprecipitation assay. Moreover, co-transfection of MyoD and Tead2 intron reporter constructs into 10T1/2 cells activated reporter activity in a dose-dependent manner. This activation was greatly reduced when the two E-boxes were mutated. Our data suggest a novel MyoD-Tead2-Fgfr4 pathway important for effective muscle regeneration.

Animals↗

PubMed2005

Putative tumor suppressor RASSF1 interactive protein and cell death inducer C19ORF5 is a DNA binding protein.

C19ORF5 is a homologue of microtubule-associated protein MAP1B that interacts with natural paclitaxel-like microtubule stabilizer and candidate tumor suppressor RASSF1A. Although normally distributed throughout the cytosol, C19ORF5 specifically associates with microtubules stabilized by paclitaxel or RASSF1A. At sufficiently high concentrations, C19ORF5 causes mitochondrial aggregation and genome destruction (MAGD). The accumulation on hyperstabilized microtubules coupled to MAGD has been proposed to mediate tumor suppression by the taxoid drug family and RASSF1A. Here, we show that the C-terminus of C19ORF5 (C19ORF5C) interacts with mitochondria-associated DNA binding protein, LRPPRC, in liver cells. Like LRPPRC, C19ORF5 also binds DNA with an affinity and specificity sufficient to be of utility in DNA affinity chromatography to purify homogeneous recombinant C19ORF5C from bacterial extracts. Homogeneous C19ORF5 exhibited no intrinsic DNase activity. Deletion mutagenesis indicated that C19ORF5 selectively binds double stranded DNA through its microtubule binding domain. These results suggest C19ORF5 as a DNA binding protein similar to microtubule-associated proteins tau and MAP2.

Animals↗

PubMed2005

Distinct structural domains within C19ORF5 support association with stabilized microtubules and mitochondrial aggregation and genome destruction.

C19ORF5 is a sequence homologue of microtubule-associated proteins MAP1A/MAP1B of unknown function, except for its association with mitochondria-associated proteins and the paclitaxel-like microtubule stabilizer and candidate tumor suppressor RASSF1A. Here, we show that when overexpressed in mammalian cells the recombinant 393-amino acid residue COOH terminus of C19ORF5 (C19ORF5C) exhibited four types of distribution patterns proportional to expression level. Although normally distributed throughout the cytosol without microtubular association, C19ORF5C specifically accumulated on stabilized microtubules in paclitaxel-treated cells and interacted directly with paclitaxel-stabilized microtubules in vitro. The native 113-kDa full-length C19ORF5 and a shorter 56-kDa form similarly associated with stabilized microtubules in liver cells and stabilized microtubules from their lysates. As C19ORF5 accumulated, it appeared on mitochondria and progressively induced distinct perinuclear aggregates of mitochondria. C19ORF5 overlapped with cytochrome c-deficient mitochondria with reduced membrane potential. Mitochondrial aggregation resulted in gross degradation of DNA, a cell death-related process we refer to as mitochondrial aggregation and genome destruction (MAGD). Deletion mutagenesis revealed that the C19ORF5 hyperstabilized microtubule-binding domain resides in a highly basic sequence of <100 residues, whereas the MAGD activity resides further downstream in a distinct 25-residue sequence (F967-A991). Our results suggest that C19ORF5 mediates communication between the microtubular cytoskeleton and mitochondria in control of cell death and defective genome destruction through distinct bifunctional structural domains. The accumulation of C19ORF5 and resultant MAGD signaled by hyperstabilized microtubules may be involved in the tumor suppression activity of RASSF1A, a natural microtubule stabilizer and interaction partner with C19ORF5, and the taxoid drug family.

Amino Acid Sequence↗

PubMed2005

Independent repression of bile acid synthesis and activation of c-Jun N-terminal kinase (JNK) by activated hepatocyte fibroblast growth factor receptor 4 (FGFR4) and bile acids.

The fibroblast growth factor (FGF) receptor complex is a regulator of adult organ homeostasis in addition to its central role in embryonic development and wound healing. FGF receptor 4 (FGFR4) is the sole FGFR receptor kinase that is significantly expressed in mature hepatocytes. Previously, we showed that mice lacking mouse FGFR4 (mR4(-/-)) exhibited elevated fecal bile acids, bile acid pool size, and expression of liver cholesterol 7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme for canonical neutral bile acid synthesis. To prove that hepatocyte FGFR4 was a negative regulator of cholesterol metabolism and bile acid synthesis independent of background, we generated transgenic mice overexpressing a constitutively active human FGFR4 (CahR4) in hepatocytes and crossed them with the FGFR4-deficient mice to generate CahR4/mR4(-/-) mice. In mice expressing active FGFR4 in liver, fecal bile acid excretion was 64%, bile acid pool size was 47%, and Cyp7a1 expression was 10-30% of wild-type mice. The repressed level of Cyp7a1 expression was resistant to induction by a high cholesterol diet relative to wild-type mice. Expression of CahR4 in mR4(-/-) mouse livers depressed bile acid synthesis below wild-type levels from the elevated levels observed in mR4(-/-). Levels of phosphorylated c-Jun N-terminal kinase (JNK), which is part of a pathway implicated in bile acid-mediated repression of synthesis, was 30% of wild-type levels in mR4(-/-) livers, whereas CahR4 livers exhibited an average 2-fold increase. However, cholate still strongly induced phospho-JNK in mR4(-/-) livers. These results confirm that hepatocyte FGFR4 regulates bile acid synthesis by repression of Cyp7a1 expression. Hepatocyte FGFR4 may contribute to the repression of bile acid synthesis through JNK signaling but is not required for activation of JNK signaling by bile acids.

Animals↗

PubMed2005

Specificity of the methylation-suppressed A isoform of candidate tumor suppressor RASSF1 for microtubule hyperstabilization is determined by cell death inducer C19ORF5.

Isoform-specific epigenetic silencing of RASSF1A (3p21.3) by promoter-specific CpG island hypermethylation occurs at high frequency in human tumors, whereas the closely related product of the same gene, RASSF1C, continues to be expressed. Both isoforms in isolation exhibit tumor suppressor properties and we show here similar cellular locations on mitochondria and microtubules, paclitaxel-like microtubule hyperstabilization, disruption of mitosis, and interaction with C19ORF5. We show both have identical but distinct sequence domains for microtubule association and hyperstabilization. C19ORF5 is a hyperstabilized microtubule-specific binding protein of which accumulation causes mitochondrial aggregation and cell death. We report herein that when A or C isoforms of RASSF1 are coexpressed with C19ORF5, the unique N-terminal sequence of RASSF1C prevents it from hyperstabilizing microtubules. This confers specificity on RASSF1A in microtubule hyperstabilization and accumulation of C19ORF5 on microtubules and could underlie a specific effect of hypermethylation-suppressed RASSF1A in tumor suppression.

Amino Acid Sequence↗

PubMed2004

Restoration of fibroblast growth factor receptor2 suppresses growth and tumorigenicity of malignant human prostate carcinoma PC-3 cells.

BACKGROUND: Fibroblast growth factors (FGFs) and their receptors (FGFRs) expedite stromal-epithelial communication in development and homeostasis of the human prostate. Loss of resident epithelial cell FGFR2IIIb that responds to stromal FGF7 and FGF10 accompanies malignant progression of both model animal and human prostate tumors. METHODS: We examined whether restoration of FGFR2IIIb by transfection in the malignant human prostate tumor PC-3 cell line restored cellular properties associated with less malignant tumors. Cell proliferation, apoptosis, and tumor cell implants were used to monitor malignant properties. Activity of FGFR2IIIb was assessed by immunoblot of FRS2 and p44/42 MAP kinase. Immunochemical analysis of pancytokeratin and lactoferrin expression was utilized to assess changes in cellular differentiation. RESULTS: Expression of FGFR2IIIb in PC-3 cells by transfection resulted in growth suppression in vitro and reduced tumor formation in vivo concurrent with increased cellular differentiation and apoptosis. CONCLUSIONS: The results indicate that restoration of FGFR2IIIb to the malignant human prostate epithelial cell prototype PC-3 restores properties associated with nonmalignant tumors and normal cells. This further suggests that epithelial cell resident, homeostasis-promoting FGFR2 may be involved in suppression of malignancy and that restoration may be a candidate for gene therapy of hormone-refractory prostate cancer.

Apoptosis↗

PubMed2004

Directionally specific paracrine communication mediated by epithelial FGF9 to stromal FGFR3 in two-compartment premalignant prostate tumors.

Tissue homeostasis in normal prostate and two-compartment nonmalignant prostate tumors depends on harmonious two-way communications between epithelial and stromal compartments. Within the fibroblast growth factor (FGF) family, signaling to an epithelial cell-specific FGF receptor (FGFR) 2IIIb-heparan sulfate complex from stromal-specific FGF7 and FGF10 delivers directionally specific instruction from stroma to epithelium without autocrine interference. Using a two-compartment transplantable prostate tumor model in which survival of stromal cells in vivo depends on epithelial cells, we show that signaling from epithelial FGF9 to stromal FGFR3 potentially mediates epithelial-to-stromal communication that also is directionally specific. FGF9 mRNA was expressed exclusively in the epithelial cells derived from well-differentiated, two-compartment Dunning R3327 rat prostate tumors. In contrast, FGFR3 was expressed at functionally significant levels only in the derived stromal cells. Competition binding and immunoprecipitation assays revealed that FGF9 only bound to an FGFR on the stromal cells. FGF9 also failed to covalently cross-link to clonal lines of stromal cells devoid of FGFR3 that expressed FGFR1 and FGFR2IIIc. Furthermore, FGF9 specifically stimulated DNA synthesis in stromal cells expressing FGFR3. These results demonstrate a directionally specific paracrine signaling from epithelial FGF9 and stromal FGFR3. Similar to the FGF7/FGF10 to FGFR2IIIb signaling from the stroma to the epithelium, the directional specificity from epithelium to stroma appears set by a combination of cell-specific expression of isoforms and cell-context specificity of FGFR isotypes for FGF.

Animals↗

PubMed2004

Chronic activity of ectopic type 1 fibroblast growth factor receptor tyrosine kinase in prostate epithelium results in hyperplasia accompanied by intraepithelial neoplasia.

BACKGROUND: Ectopic expression of fibroblast growth factor receptor 1 (FGFR1) tyrosine kinase in epithelial cells is associated with progression of prostate cancer. Ectopic expression by transfection of FGFR1 in premalignant epithelial cells from nonmalignant Dunning tumors accelerated time-dependent progression of epithelial cells to malignancy. This study was designed to test the effect of chronic androgen-dependent ectopic activity of FGFR1 in the normal adult mouse epithelium by gene targeting. MATERIALS AND METHODS: Constitutively active FGFR1 (caFGFR1) was targeted to prostate epithelial cells using the androgen-dependent probasin (PB) promoter. Prostate tissues of three strains were characterized over a period of 2 years by HE staining, immunohistochemical analyses for cytokeratin and alpha-actin, and rate of androgen-induced regeneration after castration. RESULTS: Relative to wildtype littermates, transgenic mice showed increased overall size, hyperplasia in epithelial, and, to a lesser extent, stromal compartments and nuclear atypia in epithelial cells of the prostate with increasing age. Androgen-induced regeneration after castration was enhanced at day 3 by two-fold in mice expressing ectopic caFGFR1. CONCLUSIONS: The ectopic presence and chronic activation of FGFR1 in mouse prostate epithelial cells induces progressive prostate intraepithelial neoplasia. These results confirm results suggested by the transplantable Dunning tumor and cell culture models that, in contrast to homeostasis-promoting resident FGFR2, chronic ectopic FGFR1 kinase activity in the epithelium disrupts homeostasis between stroma and epithelium. Although insufficient alone, it may cooperate with other oncogenic changes to promote epithelial cells down the path to malignancy.

Actins↗

PubMed2004

Improved production of recombinant fibroblast growth factor 7 (FGF7/KGF) from bacteria in high magnesium chloride.

Because of specificity for both heparin/heparan sulfate and the receptor complex on epithelial cells relative to other fibroblast growth factor (FGF) homologues, there is considerable interest in clinical and commercial applications of FGF7 (also called keratinocyte growth factor or KGF) that require large quantities at reasonable cost. Production of recombinant FGF7 from bacteria suffers from lower yields and recovery relative to FGF1 and FGF2. Fusion of FGF7 at the N-terminus with glutathione-S-transferase (GST) followed by removal of GST by proteolysis while bound to natural ligand heparin improved the intrinsically low yields from Escherichia coli hosts to 3.2 mg per liter per OD(600), which was still only 10% of that for FGF1. Yield of the GST-FGF7 fusion product was improved to about 17 mg per liter per OD(600) in strain BL21(DE3)pLysS by inclusion of 10-100mM magnesium chloride (MgCl(2)) in the culture medium. This improved by about five times the yields of fully active 54ser-FGF7 after proteolytic excision of the GST portion from GST-FGF7 immobilized on heparin-Sepharose. This simple enhancement improves the cost-effectiveness of production of recombinant FGF7 in bacteria for clinical and commercial applications.

Culture Media↗

PubMed2003

Cooperation between ectopic FGFR1 and depression of FGFR2 in induction of prostatic intraepithelial neoplasia in the mouse prostate.

Disruption of the regulatory communication from the stroma to the epithelium mediated by the FGF7/10-FGFR2 signaling axis in the prostate and expression of ectopic FGFR1 in prostatic epithelial cells often correlate with prostate cancer progression both in human and in experimental animals. Ectopic expression of constitutively active FGFR1 mutant (caFGFR1) at low levels in prostate epithelial cells induces low- to intermediate-grade prostatic intraepithelial neoplasia (PIN) within 6-8 months and high-grade PIN in 20-25 months. Depression of the FGFR2 signaling in the prostate also disturbs homeostasis in the prostate and induces prostate hyperplasia. To study whether PIN lesions induced by the caFGFR1 were expression-level dependent, and whether expression of the caFGFR1 and depression of the FGFR2 signaling in the prostate synergistically disturbed prostate homeostasis, we generated two new strains of ARR2PBi-caFGFR1 transgenic mice, which highly expressed caFGFR1 in prostatic epithelial cells. The mice were crossed with KDNR mice to generate ARR2PBi-caFGFR1/KDNR bigenic mice. The ARR2PBi-caFGFR1 mice developed high-grade PIN within 8 months, which was significantly faster than the mice expressing caFGFR1 at low levels. In addition, depression of the FGFR2 signaling clearly promoted perturbation of cellular homeostasis induced by the caFGFR1. The results demonstrated that the PIN development in caFGFR1 transgenic mice was caFGFR1 dosage-dependent, and indicated that the ectopic FGFR1 and the resident FGFR2 in epithelial cells had opposite impacts on intercompartmental homeostasis in the prostate. The bigenic mice provide a model with cooperative aberrations in the fibroblast growth factor signaling axis for evaluation of tumor-initiating events in prostate tumorigenesis.

Animals↗

PubMed2003

Stromal cell heterogeneity in fibroblast growth factor-mediated stromal-epithelial cell cross-talk in premalignant prostate tumors.

Homeostasis of normal prostate and two-compartment nonmalignant prostate tumors is dependent on two-way communication between epithelial and stromal compartments. Independence of epithelial cells on controlling instructions from stroma is a hallmark of extremely malignant epithelial cell tumors. To better understand the evolution of stromal independence during malignant progression, we performed a clonal analysis of stromal cells derived from a well-defined model of two-way stromal-epithelial cell communication that loses response to stroma during prostate tumor progression. Directionally specific signaling from stroma to epithelium contributes to homeostasis between the two compartments. Stromal cells were characterized in respect to expression and activity of isotypes of the fibroblast growth factor (FGF) family of ligands and receptors in addition to morphology and cytoskeletal markers. One stromal subtype (DTS1) exhibited a fibroblast-like morphology and did not display smooth muscle cell (SMC) alpha-actin. The other (DTS2) exhibited SMC alpha-actin and an SMC-like morphology in vitro. Both subtypes expressed FGF7 and equally low levels of FGFR2IIIc mRNA, whereas fibroblast growth factor receptor (FGFR) 1 predominated in DTS1 cells. DTS1 cells also expressed FGF10 and no detectable FGFR3, whereas the absence of FGF10 and presence of FGFR3 distinguished DTS2 cells. Epithelial cell-derived FGF9 bound to FGFR and stimulated growth of specifically FGFR3-positive DTS2 cells, not the FGFR3-negative DTS1 cells. These results demonstrate stromal cell heterogeneity in signal reception of FGF from epithelium. This correlated with potential heterogeneity in the response back to epithelial cells. Epithelium-dependent control of a stromal cell phenotype within a tumor may be a determinant of whether tumors remain in nonmalignant homeostasis or progress to malignancy.

Adenocarcinoma↗