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Joseph Locker

Publications and source records attributed to Joseph Locker.

15 recordsLinked to original sources
PubMed2006

Loss of Nkx2.8 deregulates progenitor cells in the large airways and leads to dysplasia.

Nkx2.8, a homeodomain transcription factor, has been characterized in liver cancer and in the developing central nervous system. We now show that this factor is also expressed in the lung, where it localizes in adults to a discrete population of tracheobronchial basal cells. To target the mouse gene, the first exon was replaced by a LacZ marker gene joined to the intact 5'-untranslated region. Marker expression was observed throughout the lower respiratory tract, beginning on E11 in a few cells of the distal lung buds. The region of expression then spread upward. By neonatal day 1, expression was greatest in the large airways and the Nkx2.8-/- mice exhibited generalized tracheobronchial hyperplasia. Bromodeoxyuridine (BrdUrd) labeling studies showed that a higher rate of bronchial cell proliferation persisted at 6 to 8 months. In adults, Nkx2.8 marker expression decreased with progressive differentiation into ciliated and secretory cells. The cell localizations and patterns of coexpression with BrdUrd and differentiation markers suggest a progenitor relationship: the cells that most strongly express Nkx2.8 seem to function as tracheobronchial stem cells. Moreover, Nkx2.8 acts to limit the number of these progenitor cells because the marker-expressing population was greatly expanded in Nkx2.8-/- mice. Increased proliferation and an altered progenitor relationship caused progressive bronchial pathology, which manifested as widespread dysplasia in the large airways of 1-year-old Nkx2.8-/- mice.

Alleles↗
PubMed2006

Dissection of Tbx1 and Fgf interactions in mouse models of 22q11DS suggests functional redundancy.

The 22q11 deletion syndrome (22q11DS) is characterized by abnormal development of the pharyngeal apparatus. Mouse genetic studies have identified Tbx1 as a key gene in the etiology of the syndrome, in part, via interaction with the fibroblast growth factor (Fgf) genes. Three murine Fgfs, Fgf3, Fgf8 and Fgf10 are coexpressed in different combinations with Tbx1. They are all strongly downregulated in Tbx1-/- embryos, implicating epistatic interactions. Supporting this, Tbx1 and Fgf8 have been shown to genetically interact in the development of the fourth pharyngeal arch artery (PAA) and Fgf10 was identified to be a direct downstream target of Tbx1. To dissect the epistatic relationships of these genes during embryonic development and the molecular pathogenesis of the Tbx1 mutant phenotype, we generated Fgf10+/-;Tbx1+/- and Fgf3-/-;Tbx1+/- mice. Despite strong hypotheses that Fgf10 is the key gene downstream of Tbx1 in the development of the anterior heart field, we do not find evidence for genetic interaction between Tbx1 and Fgf10. Also, the Fgf3-/-;Tbx1+/- mutant mice do not show an additive phenotype. Furthermore, more severe defects do not occur in Fgf8+/-;Tbx1+/- mutants by crossing in the Fgf3 null allele. There is a possible additive effect only in PAA remodeling in the Fgf10+/-;Tbx1+/-;Fgf8+/- embryos. Our findings underscore the importance of potential functional redundancy with additional Fgfs in the development of the pharyngeal apparatus and cardiovascular system via Tbx1. This redundancy should be considered when looking at individual FGF genes as modifiers of 22q11DS.

Animals↗
PubMed2006

Characterization of distant enhancers and promoters in the albumin-alpha-fetoprotein locus during active and silenced expression.

The albumin and alpha-fetoprotein genes are adjacent and express closely related serum proteins. Both genes are strongly expressed in fetal liver, primarily through activation by distant enhancers, but the AFP gene selectively undergoes developmental silencing. We used chromatin immunoprecipitation to study enhancers and promoters during active and silenced gene expression. In adult phenotype cells, the silenced AFP gene was actively repressed at the promoter and two proximal enhancers, characterized by the absence of coactivators and acetylated histone 4, and the presence of corepressors and K9-methylated histone 3. Specific transcription factors, TBP, and RNA polymerase II were all detected on both active and silenced genes, indicating that both states were actively regulated. Surprisingly, promoter-specific factors were also detected on enhancers, especially with reduced chromatin shearing. Under these conditions, an enhancer-specific factor was also detected on the albumin promoter. Association of promoter- and enhancer-specific factors was confirmed by sequential immunoprecipitation. Because no binding was detected on intervening segments, these promoter-enhancer associations suggest looping.

Albumins↗
PubMed2006

ErbB3-dependent motility and intravasation in breast cancer metastasis.

A better understanding of how epidermal growth factor receptor family members (ErbBs) contribute to metastasis is important for evaluating ErbB-directed therapies. Activation of ErbB3/ErbB2 heterodimers can affect both proliferation and motility. We find that increasing ErbB3-dependent signaling in orthotopic injection models of breast cancer can enhance intravasation and lung metastasis with no effect on primary tumor growth or microvessel density. Enhanced metastatic ability due to increased expression of ErbB2 or ErbB3 correlated with stronger chemotaxis and invasion responses to heregulin beta1. Suppression of ErbB3 expression reduced both intravasation and metastasis. A human breast cancer tumor tissue microarray showed a significant association between ErbB3 and ErbB2 expression and metastasis independent of tumor size. These results indicate that ErbB3-dependent signaling through ErbB3/ErbB2 heterodimers can contribute to metastasis through enhancing tumor cell invasion and intravasation in vivo and that ErbB-directed therapies may be useful for the inhibition of invasion independent of effects on tumor growth.

Animals↗
PubMed2006

Differential tissue and subcellular expressionof ERM proteins in normal and malignant tissues: cytoplasmic ezrin expression has prognostic signficance for head and neck squamous cell carcinoma.

BACKGROUND: Members of the ezrin-radixin-moesin (ERM) protein family regulate cellular shape, motility, and proliferation and potentially influence ability to metastasize. We investigated the correlation between ERM subcellular localization and survival in patients with squamous cell carcinoma (SCC) METHODS: Tissue microarrays (TMAs) were constructed from paraffin-embedded tissue. TMA sections were evaluated for ERM protein expression immunohistochemically. The results were compared across clinical and histopathologic variables RESULTS: ERM staining results for 47 patients showed that cytoplasmic ERM expression was prevalent in tumors (>92%). Whereas ezrin and moesin also localized to the membrane, only willin was found in the nucleus of tumors. Multivariable Cox regression analysis demonstrated that strong cytoplasmic ezrin expression was independently associated with poorer survival (p = .04, hazard ratio 1.82) CONCLUSIONS: Both level of expression and subcellular localization of ERM proteins may be important indicators of clinical outcome in SCC. This pilot study justifies the need for an expanded validation study of ERM proteins and clinical outcome.

Adult↗
PubMed2005

Gadd45beta is induced through a CAR-dependent, TNF-independent pathway in murine liver hyperplasia.

We previously observed that Gadd45/MyD118, a member of the Gadd45 family of inducible factors, showed the strongest immediate-early induction common to two distinctive proliferation responses of the liver: (1) regeneration induced by surgical partial hepatectomy and (2) hyperplasia induced by the primary mitogen TCPOBOP, a ligand of the constitutive androstane receptor (CAR). Gadd45 is known to be stimulated by nuclear factor (NF) B, which is activated by tumor necrosis factor alpha (TNF) in the early response to partial hepatectomy. We therefore investigated whether TNF and NFB also stimulated Gadd45 as part of the response to CAR ligands, or whether activation occurred by an alternative pathway. TCPOBOP effects were characterized in three mouse genotypes: wild-type, TNFR1-/-, and TNFR1-/-TNFR2-/-. The results showed that TCPOBOP did not activate NFB in any of the mice, but a strong induction of Gadd45 messenger RNA was observed in all three genotypes, where TCPOBOP also induced CyP2b10, a classical target gene of activated CAR, and cyclin D1, a proliferation linked gene. Thus, the absence of TNFR signaling and induction of NFB did not impair CAR-mediated gene induction. Moreover, hepatocyte proliferation was strongly induced, and at significantly higher levels than wild type, in both TNFR1-/- and TNFR1-/-TNFR2-/- mice. Further studies evaluated TCPOBOP-induced gene expression in CAR-/- mice, by microarray expression profiling and Northern blot. The induced changes in gene expression, including the stimulation of Gadd45, were almost completely abolished--hence all were mediated via CAR activation. In conclusion, in the liver, Gadd45 can be induced by a distinctive pathway that requires CAR and is independent of TNF-NFB. The greater induction of proliferation in TNFR-null mice suggests negative cross-talk between the CAR and TNF-NFB controls that regulate proliferation.

Animals↗
PubMed2005

Rybp/DEDAF is required for early postimplantation and for central nervous system development.

The Rybp/DEDAF protein has been implicated in both transcriptional regulation and apoptotic signaling, but its precise molecular function is unclear. To determine the physiological role of Rybp, we analyzed its expression during mouse development and generated mice carrying a targeted deletion of Rybp using homologous recombination in embryonic stem cells. Rybp was found to be broadly expressed during embryogenesis and was particularly abundant in extraembryonic tissues, including trophoblast giant cells. Consistent with this result, rybp homozygous null embryos exhibited lethality at the early postimplantation stage. At this time, Rybp was essential for survival of the embryo, for the establishment of functional extraembryonic structures, and for the execution of full decidualization. Through the use of a chimeric approach, the embryonic lethal phenotype was circumvented and a role for Rybp in central nervous system development was uncovered. Specifically, the presence of Rybp-deficient cells resulted in marked forebrain overgrowth and in localized regions of disrupted neural tube closure. Functions for Rybp in the brain also were supported by the finding of exencephaly in about 15% of rybp heterozygous mutant embryos, and by Rybp's distinct neural expression pattern. Together, these findings support critical roles for Rybp at multiple stages of mouse embryogenesis.

Animals↗
PubMed2004

Aging does not reduce the hepatocyte proliferative response of mice to the primary mitogen TCPOBOP.

It has been shown that the magnitude of DNA synthesis and the time at which maximal DNA synthesis occurs after two-thirds partial hepatectomy (PH) is greatly reduced in the liver of aged rodents compared to young animals. This reduction could represent an intrinsic defect in proliferation or a more specialized change in the response to PH. We therefore evaluated the proliferative capacity of hepatocytes in aged animals, following treatment with primary liver mitogens. We show that treatment of 12-month-old CD-1 mice with the hepatomitogen 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) caused an increase in hepatocyte proliferation similar to that seen in young (8-week-old) mice. The labeling index was 82% in the livers of aged mice versus 76% in young animals. Histological observation demonstrated that the number of hepatocytes entering mitoses was similar in both groups; the mitotic indices were 2.5 per thousand and 2.7 per thousand, respectively. Additional experiments showed that the timing of DNA synthesis and M phase were nearly identical in both aged and young mice. Stimulation of hepatocyte DNA synthesis was associated with increased expression of several cell cycle-associated proteins (cyclin D1, cyclin A, cyclin B1, E2F, pRb, and p107); all were comparable in aged mice and young mice. TCPOBOP treatment also increased expression of the Forkhead Box transcription factor m1b (Foxm1b) to a similar degree in both groups. In conclusion, hepatocytes retain their proliferative capacity in old age despite impaired liver regeneration. These findings suggest that therapeutic use of mitogens would alleviate the reduction in hepatocyte proliferation observed in the elderly.

Aging↗
PubMed2003

Nuclear receptors orchestrate detoxification pathways.

Two nuclear receptors of xenobiotic drugs, PXR and CAR, are central regulators of detoxification enzymes. New studies extend the role of these receptors to a natural detoxification process. They coordinate induction of proteins for storage, glucuronidation, and canalicular transport of bilirubin.

Animals↗
PubMed2003

A common set of immediate-early response genes in liver regeneration and hyperplasia.

Partial hepatectomy (PH) and some tumor-promoting agents stimulate hepatocyte cell proliferation, but each treatment acts through distinct transcription factors. We compared mouse immediate-early gene expression changes after PH with those induced by 1,4-bis[2-(3,5-dichoropyridyloxy)]benzene (TCPOBOP), a tumor-promoting liver mitogen. PH activates nuclear factor kappa B (NF-kappa B) and Stat3, whereas TCPOBOP is a ligand for the nuclear receptor, constitutive androstane receptor (CAR). RNA from 1 and 3 hours after each treatment was hybridized to a 9,000 complementary DNA (cDNA) microarray. Of about 6,000 messenger RNAs that had detectable expression, 127 showed reproducible up-regulation or down-regulation at a significant level. The TCPOBOP response was more discrete than the PH response; they amounted to 1% and 1.9% of positive hybridizations, respectively. Twenty-three genes were regulated only by TCPOBOP, 57 only by PH, and 59 by both treatments. More detailed analysis defined 16 clusters with common patterns of expression. These patterns and quantification of hybridization levels on the array were confirmed by Northern blots. TCPOBOP selectively activated expression of a number of detoxification enzymes. In conclusion, the genes that were regulated by both treatments suggest down-regulation of apoptosis, altered signal transduction, and early biogenesis of critical cell components.

Animals↗
PubMed2003

Indolent mantle cell lymphoma with nodal involvement and mutated immunoglobulin heavy chain genes.

Mantle cell lymphoma (MCL) is typically considered an aggressive but incurable neoplasm composed of cyclin D1+ monoclonal B-cells with a t(11;14)(q13;q32) and usually unmutated immunoglobulin (Ig) genes. Although it has been suggested that a more indolent leukemic disorder exists with the same phenotype and genotype but with mutated Ig genes, others have considered these cases to be variants of chronic lymphocytic leukemia. We present a case of an indolent MCL that was documented with cyclin D1 expression in a lymph node biopsy performed more than 12 years ago. The patient has peripheral blood involvement with a lymphocyte count in the reference range, variable thrombocytopenia, and minimal adenopathy but is otherwise well, never having received any antineoplastic therapy. Study of peripheral blood samples from 2002 revealed a CD5-variable B-cell monoclonal proliferation with a t(11;14)(q13;q32) plus other karyotypic abnormalities, positive fluorescence in situ hybridization studies for the CCND1/IgH translocation, and clonal Ig gene rearrangement with mutated Ig genes (95.7% homology to VH 4-31). The subtle but diagnostic lymph node biopsy in this case helps to further support that an indolent t(11;14) monoclonal lymphocytosis with mutated Ig genes can represent an MCL variant rather than chronic lymphocytic leukemia.

Adult↗
PubMed2002

Definition and prediction of the full range of transcription factor binding sites--the hepatocyte nuclear factor 1 dimeric site.

In animals, transcription factor binding sites are hard to recognize because of their extensive variation. We therefore characterized the general relationship between a specific protein-binding site and its DNA sequence and used this relationship to generate a predictive algorithm for searching other DNA sequences. The experimental process was defined by studying hepatocyte nuclear factor 1 (HNF1), which binds DNA as a dimer on two inverted-repeat 7-bp half sites separated by one base. The binding model was based on the equivalence of the two half sites, which was confirmed in examples where specific modified sites were compared. Binding competition analysis was used to determine the effects of substitution of all four bases at each position in the half site. From these data, a weighted half-site matrix was generated and the full site was evaluated as the sum of two half-site scores. This process accurately predicted even weak binding sites that were significantly different from the consensus sequence. The predictions also showed a direct correlation with measured protein binding.

Algorithms↗
PubMed2002

Interferon-beta activates multiple signaling cascades in primary human microglia.

Microglia, the resident brain macrophages, are the principal cells involved in the regulation of inflammatory and antimicrobial responses in the CNS. Interferon-beta (IFNbeta) is an antiviral cytokine induced by viral infection or following non-specific inflammatory challenges of the CNS. Because of the well-known anti-inflammatory properties of IFNbeta, it is also used to treat multiple sclerosis, an inflammatory CNS disease. Despite the importance of IFNbeta signaling in CNS cells, little has been studied, particularly in microglia. In this report, we investigated the molecular mechanisms underlying IFNbeta-induced beta-chemokine expression in primary human fetal microglia. Multiple signaling cascades are activated in microglia by IFNbeta, including nuclear factor-kappaB (NF-kappaB), activator protein-1 (AP-1) and Jak/Stat. IFNbeta induced IkappaBalpha degradation and NF-kappaB (p65:p50) DNA binding. Inhibition of NF-kappaB by either adenoviral transduction of a super repressor IkappaBalpha, or an antioxidant inhibitor of NF-kappaB reduced expression of the beta-chemokines, regulated upon activation, normal T-cell expressed and secreted (RANTES) and macrophage inflammatory protein (MIP)-1beta. IFNbeta also induced phosphorylation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase, and the MAP kinase kinase 1 (MEK1) inhibitor PD98059 dose-dependently inhibited beta-chemokine mRNA and protein expression. PD98059 did not inhibit NF-kappaB binding, demonstrating that ERK was not responsible for NF-kappaB activation. Two downstream targets of ERK were identified in microglia: AP-1 and Stat1. IFNbeta induced AP-1 nuclear binding activity in microglia and this was suppressed by PD98059. Additionally, IFNbeta induced Stat1 phosphorylation at both tyrosine 701 (Y701) and serine 727 (S727) residues. S727 phosphorylation of Stat1, which is known to be required for maximal transcriptional activation, was inhibited by PD98059. Our results demonstrating multiple signaling cascades initiated by IFNbeta in primary human microglia are novel and have implications for inflammatory and infectious diseases of the CNS.

Cells, Cultured↗
PubMed2002

Regulation of alpha-fetoprotein expression by Nkx2.8.

The alpha-fetoprotein (AFP) gene is an important model of developmental gene silencing and neoplastic gene reactivation. Nkx2.8 is a divergent homeodomain factor originally cloned through its binding to the promoter-coupling element (PCE), a regulatory region upstream of the AFP promoter that mediates stimulation by distant enhancers. Nkx2.8 is the only developmentally regulated factor that has been associated with AFP gene expression. Fetoprotein transcription factor, an orphan nuclear receptor, has also been shown to bind the PCE but is not developmentally regulated. The binding specificities of both families of transcription factor were determined, and overlapping sites for each were defined in the PCE. After modification of nuclear extract and gel shift analysis procedures, Nkx2.8 was identified in six AFP-positive cell lines. Transient-transfection analysis did not show transcriptional stimulation by Nkx2.8 or other active NK2 factors, which only interfered with gene expression. However, two sets of analysis demonstrated the relationship of Nkx2.8 to AFP expression: chromatin immunoprecipitation demonstrated that Nkx2.8 bound to the active AFP promoter, and antisense inhibition of Nkx2.8 mRNA translation selectively reduced expression of both the endogenous human AFP gene and transfected reporters containing the rat AFP promoter.

Animals↗