ABSTRACT
To study the effect of melanogenesis on HIF-1α expression and attendant pathways, we used stable human and hamster melanoma cell lines in which the amelanotic vs. melanotic phenotypes are dependent upon the concentration of melanogenesis precursors in the culture media. The induction of melanin pigmentation led to significant up-regulation of HIF-1α, but not HIF-2α, protein in melanized cells for both lines. Similar upregulation of nuclear HIF-1α was observed in excisions of advanced melanotic vs. amelanotic melanomas. In cultured cells, melanogenesis also significantly stimulated expression of classical HIF-1-dependent target genes involved in angiogenesis and cellular metabolism, including glucose metabolism and stimulation of activity of key enzymes in the glycolytic pathway. Several other stress related genes containing putative HRE consensus sites were also upregulated by melanogenesis, concurrently with modulation of expression of HIF-1-independent genes encoding for steroidogenic enzymes, cytokines and growth factors. Immunohistochemical studies using a large panel of pigmented lesions revealed that higher levels of HIF-1α and GLUT-1 were detected in advanced melanomas in comparison to melanocytic nevi or thin melanomas localized to the skin. However, the effects on overall or disease free survival in melanoma patients were modest or absent for GLUT-1 or for HIF-1α, respectively. In conclusion, induction of the melanogenic pathway leads to robust upregulation of HIF-1-dependent and independent pathways in cultured melanoma cells, suggesting a key role for melanogenesis in regulation of cellular metabolism.
Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Melanins/biosynthesis , Melanoma/metabolism , Skin Neoplasms/metabolism , Adult , Aged , Aged, 80 and over , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Line, Tumor , Cricetinae , Disease Progression , Female , Glucose Transporter Type 1/metabolism , Glycolysis , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Male , Melanocytes/metabolism , Melanoma/etiology , Melanoma/genetics , Melanoma, Amelanotic/etiology , Melanoma, Amelanotic/genetics , Melanoma, Amelanotic/metabolism , Middle Aged , Models, Biological , Signal Transduction , Skin Neoplasms/etiology , Skin Neoplasms/genetics , Up-Regulation , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , Young AdultABSTRACT
The role of the hypoxic response during metastasis was analysed in migrating border cells of the Drosophila ovary. Acute exposure to 1% O(2) delayed or blocked border cell migration (BCM), whereas prolonged exposure resulted in the first documented accelerated BCM phenotype. Similarly, manipulating the expression levels of sima, the Drosophila hypoxia-inducible factor (HIF)-1alpha ortholog, revealed that Sima can either block or restore BCM in a dose-dependent manner. In contrast, over-expression of Vhl (Drosophila von Hippel-Lindau) generated a range of phenotypes, including blocked, delayed and accelerated BCM, whereas over-expression of hph (Drosophila HIF prolyl hydroxylase) only accelerated BCM. Mosaic clone analysis of sima or tango (HIF-1beta ortholog) mutants revealed that cells lacking Hif-1 transcriptional activity were preferentially detected in the leading cell position of the cluster, resulting in either a delay or acceleration of BCM. Moreover, in sima mutant cell clones, there was reduced expression of nuclear slow border cells (Slbo) and basolateral DE-cadherin, proteins essential for proper BCM. These results show that Sima levels define the rate of BCM in part through regulation of Slbo and DE-cadherin, and suggest that dynamic regulation of Hif-1 activity is necessary to maintain invasive potential of migrating epithelial cells.
Subject(s)
Cell Hypoxia/physiology , Cell Movement/physiology , Drosophila/cytology , Hypoxia-Inducible Factor 1/physiology , Ovary/pathology , Animals , Dose-Response Relationship, Drug , Drosophila/genetics , Drosophila/physiology , Female , Gene Expression Regulation, Neoplastic , Neoplasm Invasiveness/physiopathologyABSTRACT
To identify functions of the IKKalpha subunit of IkappaB kinase that require catalytic activity, we generated an Ikkalpha(AA) knockin allele containing alanines instead of serines in the activation loop. Ikkalpha(AA/AA) mice are healthy and fertile, but females display a severe lactation defect due to impaired proliferation of mammary epithelial cells. IKKalpha activity is required for NF-kappaB activation in mammary epithelial cells during pregnancy and in response to RANK ligand but not TNFalpha. IKKalpha and NF-kappaB activation are also required for optimal cyclin D1 induction. Defective RANK signaling or cyclin D1 expression results in the same phenotypic effect as the Ikkalpha(AA) mutation, which is completely suppressed by a mammary specific cyclin D1 transgene. Thus, IKKalpha is a critical intermediate in a pathway that controls mammary epithelial proliferation in response to RANK signaling via cyclin D1.
Subject(s)
Cyclin D1/metabolism , Epithelial Cells/metabolism , Glycoproteins/metabolism , Mammary Glands, Animal/growth & development , Protein Serine-Threonine Kinases/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Signal Transduction , Animals , Carrier Proteins/genetics , Carrier Proteins/metabolism , Caseins/genetics , Caseins/metabolism , Cells, Cultured , Cyclin D1/genetics , Female , Fibroblasts/drug effects , Fibroblasts/metabolism , Gene Expression Regulation , Glycoproteins/genetics , Humans , I-kappa B Kinase , Lactation/physiology , Mammary Glands, Animal/cytology , Mammary Glands, Animal/metabolism , Mammary Glands, Animal/transplantation , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Mice , Mice, Transgenic , NF-kappa B/metabolism , Osteoprotegerin , Pregnancy , Protein Serine-Threonine Kinases/genetics , RANK Ligand , Receptor Activator of Nuclear Factor-kappa B , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Tumor Necrosis Factor , Tissue Transplantation , Transgenes , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
The ability to respond to differential levels of oxygen is important to all respiring cells. The response to oxygen deficiency, or hypoxia, takes many forms and ranges from systemic adaptations to those that are cell autonomous. Perhaps the most ancient of the cell-autonomous adaptations to hypoxia is a metabolic one: the Pasteur effect, which includes decreased oxidative phosphorylation and an increase in anaerobic fermentation. Because anaerobic fermentation produces far less ATP than oxidative phosphorylation per molecule of glucose, increased activity of the glycolytic pathway is necessary to maintain free ATP levels in the hypoxic cell. Here, we present genetic and biochemical evidence that, in mammalian cells, this metabolic switch is regulated by the transcription factor HIF-1. As a result, cells lacking HIF-1alpha exhibit decreased growth rates during hypoxia, as well as decreased levels of lactic acid production and decreased acidosis. We show that this decrease in glycolytic capacity results in dramatically lowered free ATP levels in HIF-1alpha-deficient hypoxic cells. Thus, HIF-1 activation is an essential control element of the metabolic state during hypoxia; this requirement has important implications for the regulation of cell growth during development, angiogenesis, and vascular injury.
Subject(s)
DNA-Binding Proteins/physiology , Nuclear Proteins/physiology , Adaptation, Physiological , Animals , Cell Hypoxia/physiology , Cell Line , Energy Metabolism , Fibroblasts , Hypoxia-Inducible Factor 1 , Hypoxia-Inducible Factor 1, alpha Subunit , Mice , Oxygen/metabolism , Transcription Factors/physiologyABSTRACT
Deletion of the transcription factor CCAAT/enhancer binding protein (C/EBP)beta results in a severe inhibition of lobuloalveolar development in the mouse mammary gland. Because progesterone receptor (PR) is requisite for alveolar development, the expression of PR was investigated in C/EBPbeta-/- mice. Unexpectedly, the number of PR-positive cells, as well as the levels of PR mRNA, were elevated 3-fold in the mammary glands of C/EBPbeta-/- mice. Furthermore, in contrast to wild-type nulliparous mice, in which PR distribution shifted from a uniform to nonuniform pattern between 8-12 weeks of age, C/EBPbeta-/- mice exhibited uniform PR distribution throughout all stages of mammary development analyzed. No change in C/EBPbeta mRNA levels was observed in the mammary glands of PR-/- mice, suggesting that PR acts in a pathway either in parallel to or downstream of C/EBPbeta. The overexpression and disrupted cellular distribution of PR in C/EBPbeta-/- mice were coincident with a striking 10-fold decrease in cell proliferation after acute steroid hormone treatment, assayed by incorporation of bromodeoxyuridine. In wild-type mice, PR and bromodeoxyuridine-positive cells were adjacent to each other and rarely colocalized. No differences in the level or pattern of PR expression were observed in the uterus, suggesting that C/EBPbeta influences PR in a mammary-specific fashion. Together, these data suggest that C/EBPbeta may control cell fate decisions in the mammary gland through the appropriate temporal and spatial expression of molecular markers, such as PR, that induce the proliferation of alveolar progenitor cells via juxtacrine mechanisms.
Subject(s)
DNA-Binding Proteins/physiology , Gene Expression Regulation, Developmental , Mammary Glands, Animal/growth & development , Nuclear Proteins/physiology , Receptors, Progesterone/metabolism , Animals , Biomarkers , CCAAT-Enhancer-Binding Proteins , Cell Division/drug effects , Cell Lineage , DNA Replication/drug effects , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Epithelial Cells/cytology , Epithelial Cells/metabolism , Female , Fluorescent Antibody Technique, Indirect , Gene Deletion , In Situ Hybridization , Mammary Glands, Animal/cytology , Mammary Glands, Animal/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Nuclear Proteins/deficiency , Nuclear Proteins/genetics , RNA, Messenger/biosynthesisABSTRACT
The CCAAT/enhancer binding proteins (C/EBPs) are differentially expressed throughout mammary gland development and interact with binding sites within the promoter of a milk protein gene, beta-casein. The specific roles of C/EBPbeta and C/EBPalpha in mouse mammary gland development and differentiation have been investigated in mice that carry targeted deletions of these genes. C/EBPbeta-/- virgin mice exhibited cystic, enlarged mammary ducts with decreased secondary branching. Transplantation of C/EBPbeta-/- mammary epithelium into the cleared mammary fat pads of nude mice confirmed that this defect in ductal morphogenesis was intrinsic to the epithelium. When treated with estrogen/progesterone (E+P) to simulate pregnancy, C/EBPbeta-/- mammary glands displayed only limited lobuloalveolar development and ductal side branching. Primary mammary epithelial cells obtained from E+P-treated C/EBPbeta-/- mice that were cultured on extracellular matrix gels did not functionally differentiate in response to lactogenic hormones despite their organization into three-dimensional structures. Expression of beta-casein protein was inhibited 85%-100% and whey acidic protein (WAP) was undetectable. In contrast, no detectable alterations in mammary development or beta-casein expression were observed in mammary outgrowths derived from newborn C/EBPalpha-/- mammary epithelium transplanted into the cleared mammary fat pads of syngeneic hosts. These results demonstrate that C/EBPbeta, but not C/EBPalpha, is required for ductal morphogenesis, lobuloalveolar development, and functional differentiation of mammary epithelial cells.
