ABSTRACT
Phosphorylation of proteins on tyrosine residues is regulated by the activities of protein tyrosine kinases and protein tyrosine phosphatases. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) essentially and transiently expressed during development of the central and peripheral nervous systems. ALK has been identified as a major neuroblastoma predisposition gene and activating mutations have been identified in a subset of sporadic neuroblastoma tumors. We previously established that the mutated receptors were essentially retained in the endoplasmic reticulum/Golgi compartments due to their constitutive activity. Intriguingly we demonstrated a stronger phosphorylation for the minor pool of receptor addressed to the plasma membrane. We decided to investigate the potential involvement of tyrosine phosphatase in dephosphorylation of this intracellular pool. In this study we first showed that general inhibition of tyrosine phosphatases resulted in a dramatic increase of the tyrosine phosphorylation of the wild type but also of the mutated receptors. This increase not only required the intrinsic kinase activity of the ALK receptor but also involved the Src tyrosine kinase family. Second we provided strong evidences that the endoplasmic reticulum associated phosphatase PTP1B is key player in the control of ALK phosphorylation. Our data shed a new light on the biological significance of the basal phosphorylation levels of both wild type and mutated ALK receptors and could be essential to further understand their roles in malignancies.
Subject(s)
Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Anaplastic Lymphoma Kinase , Animals , Dimerization , Endoplasmic Reticulum/metabolism , Humans , Mice , NIH 3T3 Cells , Phosphorylation/drug effects , Protein Tyrosine Phosphatase, Non-Receptor Type 1/genetics , Receptor Protein-Tyrosine Kinases/genetics , Transfection , Tyrosine/metabolism , Vanadates/pharmacology , src-Family Kinases/metabolismABSTRACT
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in neuroblastoma, a devastating pediatric cancer of the sympathetic nervous system. Germline and somatically acquired ALK aberrations induce increased autophosphorylation, constitutive ALK activation and increased downstream signaling. Thus, ALK is a tractable therapeutic target in neuroblastoma, likely to be susceptible to both small-molecule tyrosine kinase inhibitors and therapeutic antibodies-as has been shown for other receptor tyrosine kinases in malignancies such as breast and lung cancer. Small-molecule inhibitors of ALK are currently being studied in the clinic, but common ALK mutations in neuroblastoma appear to show de novo insensitivity, arguing that complementary therapeutic approaches must be developed. We therefore hypothesized that antibody targeting of ALK may be a relevant strategy for the majority of neuroblastoma patients likely to have ALK-positive tumors. We show here that an antagonistic ALK antibody inhibits cell growth and induces in vitro antibody-dependent cellular cytotoxicity of human neuroblastoma-derived cell lines. Cytotoxicity was induced in cell lines harboring either wild type or mutated forms of ALK. Treatment of neuroblastoma cells with the dual Met/ALK inhibitor crizotinib sensitized cells to antibody-induced growth inhibition by promoting cell surface accumulation of ALK and thus increasing the accessibility of antigen for antibody binding. These data support the concept of ALK-targeted immunotherapy as a highly promising therapeutic strategy for neuroblastomas with mutated or wild-type ALK.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Neuroblastoma/immunology , Neuroblastoma/therapy , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/immunology , Anaplastic Lymphoma Kinase , Antigens, Neoplasm/genetics , Antigens, Neoplasm/immunology , Antigens, Neoplasm/metabolism , Cell Death/drug effects , Cell Death/genetics , Cell Death/immunology , Cell Line, Tumor , Cell Proliferation/drug effects , Crizotinib , Humans , Mutation/immunology , Neuroblastoma/genetics , Neuroblastoma/metabolism , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/immunology , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Pyrazoles/pharmacology , Pyridines/pharmacology , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Signal Transduction/drug effects , Signal Transduction/immunologyABSTRACT
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK), which is transiently expressed during development of the central and peripheral nervous system. ALK has been recently identified as a major neuroblastoma predisposition gene and activating mutations have also been identified in a subset of sporadic neuroblastoma tumors. Two hot spots of ALK mutations have been observed at positions F1174 and R1275. Here, we studied stably transfected cell lines expressing wild-type or F1174L- or R1275Q-mutated ALK in parallel with a neuroblastoma cell line (CLB-GE) in which the allele mutated at position F1174 is amplified. We observed that the mutated ALK variants were essentially intracellular and were largely retained in the reticulum/Golgi compartments. This localization was corroborated by a defect of N-linked glycosylation. Although the mutated receptors exhibited a constitutive activation, the minor pool of receptor addressed to the plasma membrane was much more tyrosine phosphorylated than the intracellular pool. The use of antagonist monoclonal antibodies suggested that the constitutive activity of the mutated receptors did not require the dimerization of the receptor, whereas adequate dimerization triggered by agonist monoclonal antibodies increased this activity. Finally, kinase inactivation of the mutated receptors restored maturation and cell-surface localization. Our results show that constitutive activation of ALK results in its impaired maturation and intracellular retention. Furthermore, they provide a rationale for the potential use of kinase inhibitors and antibodies in ALK-dependent tumors.
Subject(s)
Arginine , Mutation , Phenylalanine , Receptor Protein-Tyrosine Kinases/chemistry , Receptor Protein-Tyrosine Kinases/metabolism , Anaplastic Lymphoma Kinase , Animals , Cell Line, Tumor , Cell Membrane/enzymology , Endoplasmic Reticulum/enzymology , Enzyme Activation , Glycosylation , Golgi Apparatus/enzymology , Humans , Mice , Molecular Weight , NIH 3T3 Cells , Protein Folding , Protein Transport/genetics , Receptor Protein-Tyrosine Kinases/geneticsABSTRACT
Many different types of cancer originate from aberrant signaling from the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (RTK), arising through different translocation events and overexpression. Further, activating point mutations in the ALK domain have been recently reported in neuroblastoma. To characterize signaling in the context of the full-length receptor, we have examined whether ALK is able to activate Rap1 and contribute to differentiation/proliferation processes. We show that ALK activates Rap1 via the Rap1-specific guanine-nucleotide exchange factor C3G, which binds in a constitutive complex with CrkL to activated ALK. The activation of the C3G/Rap1 pathway results in neurite outgrowth of PC12 cells, which is inhibited by either overexpression of Rap1GAP or siRNA-mediated knockdown of Rap1 itself or the guanine nucleotide exchange factor C3G. Significantly, this pathway also appears to function in the regulation of proliferation of neuroblastoma cells such as SK-N-SH and SH-SY5Y, because abrogation of Rap1 activity by Rap1-specific siRNA or overexpression of Rap1GAP reduces cellular growth. These results suggest that ALK activation of Rap1 may contribute to cell proliferation and oncogenesis of neuroblastoma driven by gain-of-function mutant ALK receptors.
Subject(s)
Guanine Nucleotide-Releasing Factor 2/metabolism , Neuroblastoma/pathology , Protein-Tyrosine Kinases/metabolism , rap1 GTP-Binding Proteins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Anaplastic Lymphoma Kinase , Animals , Cell Proliferation , Cell Transformation, Neoplastic , Enzyme Activation/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , GTPase-Activating Proteins/genetics , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Guanine Nucleotide-Releasing Factor 2/deficiency , Guanine Nucleotide-Releasing Factor 2/genetics , Humans , Mice , Neurites/metabolism , Neuroblastoma/genetics , Nuclear Proteins/metabolism , PC12 Cells , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrimidines/pharmacology , Rats , Receptor Protein-Tyrosine Kinases , rap1 GTP-Binding Proteins/deficiency , rap1 GTP-Binding Proteins/geneticsABSTRACT
BACKGROUND: Malignant tumors of the testis are among the most common cancers in men between the ages of 15 and 30 years. The sensitivity of detection of known tumor markers depends upon the tumor histology and stage. In other cancers, increased serum concentrations of various angiogenic growth factors have been described as potential markers for tumor progression and metastasis. One main histological feature of testicular cancer is profound angiogenesis. DESIGN: In this study, we investigated by sensitive enzyme-linked immunosorbent assays (ELISAs) the levels of various growth and angiogenesis factors in the serum of testicular cancer patients as compared with normal control subjects. For the most profoundly increased growth factors, pleiotrophin (PTN) and fibroblast growth factor-2 (FGF-2), we furthermore analyzed tumor lysates by northern blotting, RT-PCR and ELISA. RESULTS: We demonstrate a marked elevation of average serum levels of PTN ( approximately 20-fold) and of FGF-2 ( approximately 7-fold) in patients and expression of both growth factors in tumor biopsies. To a lesser extent, vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) serum levels were increased, whereas FGF-4 and transforming growth factor-beta levels were similar to those in normal control subjects. Elevation of PTN, FGF-2, EGF and VEGF was detected in seminomatous as well as non-seminatous tumors, and even in early stages. CONCLUSIONS: PTN and FGF-2 may represent promising new diagnostic markers for testicular cancer with high sensitivity even in early-stage testicular cancer. Further studies are warranted to extend our analyses.
Subject(s)
Biomarkers, Tumor/analysis , Carrier Proteins/analysis , Cytokines/analysis , Neovascularization, Pathologic , Testicular Neoplasms/pathology , Adult , Diagnosis, Differential , Enzyme-Linked Immunosorbent Assay , Fibroblast Growth Factor 2 , Humans , Male , Nerve Growth Factors/analysis , Proto-Oncogene Proteins/analysis , Sensitivity and Specificity , Testicular Neoplasms/diagnosisABSTRACT
Pleiotrophin (PTN) is a developmentally regulated protein that has been shown to be involved in tumor growth and metastasis presumably by activating tumor angiogenesis. To clarify the potential angiogenic activity of PTN and to analyze the signaling pathways involved in this process, we used an in vitro model of Human Umbilical Vein Endothelial Cells (HUVEC). We show that PTN was mitogenic toward a variety of endothelial cells including HUVEC, stimulated HUVEC migration across a reconstituted basement membrane and induced the formation of capillary-like structures by HUVEC grown as 3D-cultures in Matrigel or collagen. The signaling pathways triggered following endothelial cell stimulation by PTN were studied by using pharmacological inhibitors of the Phosphoinositide-3 kinase (PI3K) and endothelial Nitric Oxide Synthase (eNOS), two enzymes that have been shown to be crucial in the angiogenic response to Vascular Endothelial Growth Factor (VEGF). Whereas wortmannin (a PI3K inhibitor) and L-NAME (an eNOS inhibitor) dramatically reduced HUVEC growth induced by VEGF, only the former inhibitor reduced the growth induced by PTN and to a lesser extent that stimulated by basic Fibroblast Growth Factor. Thus, our results indicate that PTN induces angiogenesis and utilizes PI3K- but not eNOS-dependent pathways for its angiogenic activity.
Subject(s)
Carrier Proteins/physiology , Cytokines/physiology , Endothelium, Vascular/physiology , Neovascularization, Pathologic , Nitric Oxide Synthase/antagonists & inhibitors , Phosphatidylinositol 3-Kinases/physiology , Androstadienes/pharmacology , Animals , Cell Division/drug effects , Cell Line , Cell Movement/drug effects , Cells, Cultured , Endothelial Growth Factors/pharmacology , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Enzyme Inhibitors , Fibroblast Growth Factor 2/pharmacology , Humans , Lymphokines/pharmacology , NG-Nitroarginine Methyl Ester/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Rats , Signal Transduction , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors , WortmanninABSTRACT
Anaplastic lymphoma kinase (ALK) is a novel neuronal orphan receptor tyrosine kinase that is essentially and transiently expressed in specific regions of the central and peripheral nervous systems, suggesting a role in its normal development and function. To determine whether ALK could play a role in neuronal differentiation, we established a model system that allowed us to mimic the normal activation of this receptor. We expressed, in PC12 cells, a chimeric protein in which the extracellular domain of the receptor was replaced by the mouse IgG 2b Fc domain. The Fc domain induced the dimerization and oligomerization of the chimeric protein leading to receptor phosphorylation and activation, thus mimicking the effect of ligand binding, whereas the wild type ALK remained as a monomeric nonphosphorylated protein. Expression of the chimera, but not that of the wild type ALK or of a kinase inactive form of the chimera, induced the differentiation of PC12 cells. Analysis of the signaling pathways involved in this process pointed to an essential role of the mitogen-activated protein kinase cascade. These results are consistent with a role for ALK in neuronal differentiation.
Subject(s)
Cell Division , MAP Kinase Signaling System , Neurons/cytology , Protein-Tyrosine Kinases/metabolism , Anaplastic Lymphoma Kinase , Animals , Base Sequence , DNA Primers , Enzyme Activation , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Humans , PC12 Cells , Phosphorylation , Protein-Tyrosine Kinases/chemistry , Rats , Receptor Protein-Tyrosine Kinases , Tyrosine/metabolismABSTRACT
BACKGROUND: Growth factors produced by tumor cells are essential for tumor expansion and may be useful in monitoring tumor progression or therapeutic efficacy if the factors are released into the circulation. In this study, we measured serum levels of pleiotrophin, a secreted heparin-binding growth and angiogenesis factor, in mice bearing human tumor xenografts to determine whether these levels reflected overall tumor burden, and we examined the relationship between tumor expression of pleiotrophin and serum levels of this factor in patients with cancer. METHODS: Pleiotrophin in serum from mice and humans was measured by use of a highly sensitive enzyme-linked immunosorbent assay. For the clinical studies, serum specimens were obtained from 193 patients with various cancers of the gastrointestinal tract and from 28 healthy control subjects. In a subset of 64 cancer patients, serum levels of pleiotrophin were measured at the time of surgery, and tumor expression of this factor was detected immunohistochemically. All P values are two-sided. RESULTS: In mice, serum pleiotrophin levels were found to increase as a function of tumor size. In humans, elevated serum pleiotrophin levels were found in patients with pancreatic cancer (n = 41; P<.0001) and colon cancer (n = 65; P = .0079) but not in patients with stomach cancer (n = 87; P =.42). A statistically significant positive association was found between elevated levels of pleiotrophin in serum drawn at the time of surgery and expression of this factor by tumors (P<.0001). In both mice and humans, serum pleiotrophin levels dropped after successful tumor removal. CONCLUSIONS: Elevated serum pleiotrophin levels can indicate the presence of tumors expressing this factor. Monitoring serum levels of pleiotrophin may prove useful in determining the pharmacologic efficacy of cytotoxic or anti-pleiotrophin therapy.
Subject(s)
Carrier Proteins/blood , Cytokines/blood , Digestive System Neoplasms/blood , Growth Substances/blood , Adult , Aged , Animals , Case-Control Studies , Enzyme-Linked Immunosorbent Assay , Female , Humans , Male , Mice , Middle Aged , Sensitivity and Specificity , Transplantation, HeterologousABSTRACT
The heparin-binding growth-associated molecule HB-GAM (also named pleiotrophin) and the retinoic acid-induced heparin-binding protein RIHB (chicken midkine) are developmentally regulated proteins forming a new family of heparin-binding molecules with putative functions during cell growth and differentiation. A direct involvement of these molecules during chondrogenesis in vivo was suggested by their patterns of expression. The putative chondrogenic activity of these molecules was investigated in vitro using micromass cultures from chicken limb bud mesenchymal cells. Exogenous HB-GAM, not RIHB, was found to enhance chondrogenesis in this system. These results provide a strong incentive for considering and further investigating the role of this protein in the control of limb cartilage differentiation.
Subject(s)
Carrier Proteins/pharmacology , Cartilage/drug effects , Cartilage/embryology , Cytokines/pharmacology , Mitogens/pharmacology , Nerve Growth Factors/pharmacology , Animals , Carrier Proteins/isolation & purification , Cell Division/drug effects , Cells, Cultured , Chick Embryo , Culture Techniques , Cytokines/isolation & purification , Limb Buds/cytology , Limb Buds/drug effects , Mesoderm/cytology , Midkine , Time FactorsABSTRACT
The heparin-binding growth-associated molecule HB-GAM (also named pleiotrophin) is a developmentally-regulated protein that belongs to a new family of heparin-binding molecules with putative functions during cell growth and differentiation. In order to study the localization of HB-GAM during chicken embryogenesis, we produced specific monoclonal antibodies to this factor. HB-GAM protein is first observed at stage 23 in the developing nervous system and later in the forming cartilage. We present an investigation of the HB-GAM mRNA expression and HB-GAM protein distribution in the developing leg by in situ hybridization and immunocytochemical studies. We focused our attention on the development of the tibia, where the HB-GAM protein appears at stage 27-28, i.e., just after the condensation of the mesodermal precursor cells of the chondrocytes. The protein then progressively accumulates in the central part of the embryonic cartilage (diaphysis). It persists until stage 42-44 in the regions where hypertrophic cartilage is being replaced by bone marrow. In contrast to the protein, the transcript is first detected at stage 26-27 and later expressed essentially in the epiphysis until stage 37. Therefore the localization of the mRNA does not parallel that of the protein and our data suggest a long half-life of the protein in the hypertrophic cartilage. In addition, the layer of stacked cells surrounding the cartilage core (usually considered as the osteoprogenitor cells) clearly expresses the HB-GAM message between stages 30-37 whereas differentiated osteoblasts do not. Furthermore, the distribution of HB-GAM protein in the osteoblast/osteoid layer suggests an involvement of this protein in early steps of osteogenesis. HB-GAM is absent from the newly formed bone.
Subject(s)
Carrier Proteins/biosynthesis , Cytokines/biosynthesis , Growth Substances/biosynthesis , Hindlimb/embryology , Mitogens/biosynthesis , Nerve Tissue Proteins/biosynthesis , RNA, Messenger/metabolism , Animals , Antibodies, Monoclonal/metabolism , Carrier Proteins/immunology , Chick Embryo , Cytokines/immunology , Growth Substances/immunology , In Situ Hybridization , Mitogens/immunology , Morphogenesis , Nerve Tissue Proteins/immunologySubject(s)
Ascorbic Acid/pharmacology , Avian Proteins , Carrier Proteins/biosynthesis , Cartilage/metabolism , Cytokines , 2,2'-Dipyridyl/pharmacology , Animals , Carrier Proteins/drug effects , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cartilage/cytology , Cell Differentiation , Cells, Cultured , Chick Embryo , Extracellular Matrix/metabolism , Gene Expression , Glycerophosphates/pharmacology , Intracellular Signaling Peptides and Proteins , Midkine , Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/metabolism , Procollagen-Proline Dioxygenase/metabolism , RNA, MessengerABSTRACT
Recent data have shown that NOS-I is localized almost exclusively to the sarcolemma of fast-twitch fibers, where it probably interacts with the dystrophin-glycoprotein complex. The concentration of dystrophin-related protein at the neuromuscular junctions and the possible involvement of NO in synaptic suppression led us to investigate the presence of NOS-I at the adult motor endplates. Our data clearly show that NOS-I protein, detected by immunohistochemistry accumulates at the adult endplates. Furthermore, the absence of NOS-I protein at the denervated neuromuscular junctions suggest a neural origin of this enzyme. The putative roles of NO at the endplate are discussed.
Subject(s)
Neuromuscular Junction/enzymology , Nitric Oxide Synthase/metabolism , Animals , Denervation , Immunohistochemistry , Mice , Mice, Inbred C57BLABSTRACT
The heparin-binding protein, RIHB, is encoded by a gene that is expressed temporarily during the early period of chicken embryogenesis. We have now isolated genomic clones of the chicken RIHB gene, determined its transcription initiation sites, characterized its promoter region and established the functional activity of the RIHB gene promoter. The 5' flanking region and the beginning of the first exon revealed a structural organisation characteristic of housekeeping and growth-control-related genes. It lacked canonical TATA or CAAT boxes but contained several GC boxes. The structure of the RIHB gene is compared with those of the human and mouse midgestation kidney (MK) genes. In the coding exons, the similarities between the three genes are striking. In contrast, the structure and location of the first non-coding exon is different. Analysis of the promoter activity suggests an indirect regulation of transcription by retinoic acid and demonstrates a high degree of complexity of the regulation of the RIHB gene.
Subject(s)
Avian Proteins , Carrier Proteins/genetics , Promoter Regions, Genetic , Amino Acid Sequence , Animals , Base Sequence , Cells, Cultured , Chick Embryo , DNA, Complementary , Gene Expression Regulation/drug effects , Humans , Intracellular Signaling Peptides and Proteins , Introns , Mice , Molecular Sequence Data , Protein Biosynthesis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Tretinoin/pharmacologyABSTRACT
Retinoic acid induced heparin-binding protein (RIHB) is a highly basic, soluble polypeptide of the chick embryonic extracellular matrix. We have examined the expression and localization of RIHB during very early embryogenesis by in situ hybridization and immunohistochemistry. RIHB mRNA is very weakly detectable above background in the blastodiscs of unincubated eggs. The expression increases greatly over the first 24 hours of incubation, and is observed throughout the blastodisc in all three of the germ layers following gastrulation. As neurulation occurs, the expression becomes more restricted to certain areas, notably the ectoderm, the neural folds, and especially the notochord. After the neural tube has formed the expression in the tube itself decreases dramatically, whereas the expression in the head ectoderm and the notochord persists. After 72 hours of incubation expression remains relatively high throughout most of the embryo, with higher levels of expression in regions undergoing organogenesis and lower levels in organs which have already differentiated. RIHB protein is also weakly detectable in unincubated eggs as patches of immunoreactive material between the blastodisc and the vitelline. After 6 hours of incubation small regions of basement membrane are immunoreactive. RIHB is detected in this matrix, apparently before even fibronectin. The amount of RIHB protein increases dramatically over the first 24 hours of incubation. It is found in basement membrane separating the epiblast from the hypoblast, then later in that separating the ectoderm from the mesoderm. It is also detected surrounding individual cells, especially of the ectodermal layer. During neurulation RIHB is observed in the basement membrane surrounding the neural fold and the notochord, and in the lamina separating the ectodermal, mesodermal, and endodermal layers. Later in development, RIHB is detected in the basement membrane under the epidermis, throughout the developing limbs, and in the lamina of various developing organs, such as the eye, the pulmonary bud, the intestine, and the mesonephros. These results demonstrate that RIHB is highly expressed during the early embryonic period, by all three germ layers, and is an important and very early component of the embryonic extracellular matrix. Its very broad expression and localization argue for a more general role in development than its demonstrated weak neurotrophic activity.
Subject(s)
Avian Proteins , Carrier Proteins/biosynthesis , Central Nervous System/embryology , Chick Embryo/growth & development , Gastrula/metabolism , Animals , Cell Differentiation , Cell Movement , Central Nervous System/metabolism , Chick Embryo/metabolism , In Situ Hybridization , Intracellular Signaling Peptides and ProteinsABSTRACT
Previously, we isolated an avian protein which we named retinoic acid induced heparin binding factor (RIHB). RIHB is a 121 amino acid secreted polypeptide, rich in basic and cysteine residues (Vigny et al., Eur. J. Biochem. 186: 733-740, 1989). Northern blot analysis indicates that the RIHB gene is transiently expressed during embryogenesis (Urios et al., Biochem. Biophys. Res. Com. 175:617-624, 1991). Here we present an investigation of RIHB expression during early chicken embryogenesis by in situ hybridization and immunofluorescence studies. In the 3-day embryo (stage 20-21), the RIHB transcript is observed throughout the embryo, with the notable exception of the neural tube. At this stage the protein can be visualized in almost all of the basement membranes and around many types of cells. The localization of the RIHB protein does not strictly parallel that of its messenger. Between days 3 and 11 we focused our attention on wing development. The level of both the mRNA and protein decreases during this period but the disappearance is not uniform. The level of both the mRNA and protein decreases during this period but the disappearance is not uniform. The transcript becomes progressively restricted to epithelia and regions surrounding the forming cartilage. In contrast to the transcript, the protein accumulates in the epithelial basement membrane and, interestingly, in the central part of the embryonic cartilage (diaphysis) but not in the distal parts (epiphysis). These data are discussed in relation to the putative role(s) of RIHB in development.
Subject(s)
Avian Proteins , Carrier Proteins/analysis , Chick Embryo/chemistry , Wings, Animal/chemistry , Animals , Carrier Proteins/physiology , Cartilage/chemistry , Cartilage/embryology , Chick Embryo/growth & development , Fluorescent Antibody Technique , Gene Expression , In Situ Hybridization , Intracellular Signaling Peptides and Proteins , Morphogenesis/physiology , RNA, Messenger/analysis , Wings, Animal/embryologyABSTRACT
Retinoic acid-induced heparin-binding factor (RIHB) is a highly basic polypeptide expressed during early chick embryogenesis. We have examined the induction of RIHB by retinoic acid in chondrocytes isolated from the sterna of Day 15 chick embryos and the effects of exogenous RIHB on these cells. There is an induction of RIHB mRNA in chondrocytes which is dose dependent, with maximal levels of expression observed with concentrations of retinoic acid in the 10(-6) M range. RIHB mRNA is first observed 16 h after commencement of treatment, is maximal after 24-48 h, and is completely attenuated after 5 days. This transient pattern of expression is very similar to that of type X collagen; however, RIHB induction precedes that of type X collagen by about 24 h. The expression of both RIHB and type X collagen precedes the drop in keratan sulfate:chondroitin sulfate proteoglycan and type II collagen expression and the surge of fibronectin expression. The induction of RIHB mRNA is accompanied by an increased synthesis of the protein. No RIHB can be detected in untreated chondrocytes; however, large amounts are produced by cells treated with 5 x 10(-7) M retinoic acid. The protein is recovered mainly in the culture medium and bound to the extracellular matrix. Only a small amount can be detected in cell extracts. RIHB can be detected in the culture medium after 16-24 h and, unlike the mRNA, persists over the 5-day period examined. The effect of exogenous RIHB (purified from chick embryos) on chondrocyte proliferation and morphology was examined. When added to the culture medium in concentrations of up to 500 ng/ml RIHB had no effect on [3H]thymidine incorporation or cell morphology. Thus, RIHB is not the direct mediator of retinoic acid for these cells, but is strongly induced during the treatment.
Subject(s)
Cartilage , Fibroblast Growth Factor 2/genetics , RNA, Messenger/metabolism , Tretinoin/pharmacology , Animals , Blotting, Northern , Cartilage/cytology , Cartilage/embryology , Cartilage/metabolism , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cells, Cultured , Chick Embryo , Chromatography, High Pressure Liquid , Collagen/genetics , Collagen/metabolism , Dose-Response Relationship, Drug , Fibroblast Growth Factor 2/drug effects , Fibroblast Growth Factor 2/physiology , Fibronectins/genetics , Fibronectins/metabolism , Fluorescent Antibody Technique , Gene Expression/genetics , Immunohistochemistry , RNA, Messenger/genetics , Time FactorsABSTRACT
Trembler mouse, a Schwann cell mutation, is characterized by severe hypomyelination of peripheral nerves, high Schwann cell proliferation and the presence of a multilayered basal lamina which surrounds them. In contrast with their continuous in vivo division, mutant Schwann cells prepared from 15-day sciatic nerves display a lower proliferation rate in cell culture than normal Schwann cells. However, quiescent Trembler Schwann cells are still able to respond, as normal Schwann cells, to exogenous mitogens, such as nerve extracts and myelin-enriched fractions. In addition, both normal and Trembler Schwann cells proliferate in response to Trembler serum. Fibroblast growth factor is not the mitogenic factor which stimulates mutant Schwann cell proliferation in vivo, since it is absent in Trembler serum and poorly concentrated in Trembler adult sciatic nerves. Our results suggest that, in vivo, the serum of Trembler mouse probably contains mitogenic factors, not yet characterized, which may trigger the permanent division of mutant Schwann cells, in contrast to the quiescent state of these cells in the nerves of normal mice.
Subject(s)
Demyelinating Diseases/pathology , Myelin Sheath/pathology , Schwann Cells/pathology , Aging/physiology , Animals , Cell Division/drug effects , Cells, Cultured , Demyelinating Diseases/blood , Demyelinating Diseases/genetics , Mice , Mice, Neurologic Mutants , Mitogens/pharmacology , Myelin Sheath/chemistry , Myelin Sheath/drug effects , Schwann Cells/drug effects , Sciatic Nerve/chemistry , Tissue Extracts/pharmacologyABSTRACT
We have conducted an immunocytochemical study of fibronectin, laminin, heparan sulfate proteoglycans and nidogen-entactin during lens and neural retina regeneration in the adult newt from 0 to 60 days. In the normal eye, fibronectin was detected in the corneal stroma and Descemet's membrane, in dorsal and ventral irises and lens capsule but not in Bowman's membrane of the cornea. In normal neural retina, fibronectin was found in Bruch's and inner limiting membranes. Heparan sulfate proteoglycans gave a slight signal in both irises and the lens capsule. Nidogen-entactin distribution in the cornea was similar to that of fibronectin; it was absent from the stroma of both irises, and the signal was weak in the pigmented iris epithelium. Nidogen-entactin was not detected in the lens capsule and inner limiting membrane of the neural retina but was present in Bruch's membrane. During the first 15 days of lens regeneration, fibronectin and nidogen-entactin decreased but did not disappear from the pupillary margin of both irises, and no signal was obtained for laminin and heparan sulfate proteoglycans. From day 15 to day 60 fibronectin and nidogen-entactin increased in both irises and lens capsule. The signal for laminin was restricted to the lens capsule. Heparan sulfate proteoglycans gave a slight signal in both irises and in the lens capsule. During the first 25 days of neural retina regeneration, fibronectin was the first to appear in Bruch's membrane and the cell border of the new neuroepithelium and remained during the entire process. Laminin appeared after 41 days in the inner limiting and Bruch's membranes, but by day 50 it appeared as a weak signal only in the inner limiting membrane. Heparan sulfate proteoglycans were not detected at any of the regeneration stages studied. Nidogen-entactin was only detected in Bruch's membrane and around the cells and blood vessels of the new neural retina. Later it was detected in the inner limiting membrane but not in Bruch's membrane. Thus, the results obtained showed that extracellular matrix components do change during both lens and neural retina regeneration. These changes may play an important role during both regenerating processes.
Subject(s)
Extracellular Matrix Proteins/metabolism , Lens, Crystalline/metabolism , Lens, Crystalline/physiology , Nerve Regeneration/physiology , Retina/metabolism , Retina/physiology , Animals , Basement Membrane/metabolism , Fluorescent Antibody Technique , Membrane Glycoproteins/metabolism , Regeneration , SalamandridaeABSTRACT
The distribution of four basement membrane components: laminin (LAM), type IV collagen (Coll. IV), heparan-sulfate proteoglycan (HSPG), and entactin (ENT), was studied by immunocytochemistry in primary cultures of adult rat anterior pituitaries. In such cultures, the pituitary cells are deprived of their normal environment of adjacent cells and basement membranes (BM), and of the connectivo-vascular system of the hypophysis. In this dissociated system, pituitary cells grow as small clusters upon a monolayer of fibroblasts. LAM was found highly expressed in endocrine and in folliculo-stellate cells. Very small amounts of Coll. IV, but neither HSPG nor ENT, could be detected in endocrine cells. In contrast, in fibroblasts, very large amounts of Coll. IV, HSPG, and ENT, and a lower quantity of LAM were detected. At the ultrastructural level, the immunoreactive components present within the cells were located in the subcellular compartments involved in the elaboration of exported products. In addition to that intracellular distribution, the four constituents were observed in an extracellular matrix which appeared between the cultured cells, either as an amorphous material, or as a more or less dense reticular network, weakly stained with anti-LAM, but strongly stained with the other antibodies. Thus, the present immunocytochemical data support the implication of pituitary endocrine cells, at least for LAM secretion, in the elaboration of a novel extracellular matrix in primary cultures. In addition, a cooperation with non-endocrine cells seemed to be required for the production of the four BM components.
Subject(s)
Collagen/analysis , Heparitin Sulfate/analysis , Laminin/analysis , Membrane Glycoproteins/analysis , Pituitary Gland, Anterior/chemistry , Pituitary Gland, Anterior/cytology , Animals , Cell Membrane/chemistry , Cell Membrane/ultrastructure , Cells, Cultured , Extracellular Matrix/chemistry , Extracellular Matrix/ultrastructure , Immunohistochemistry , Male , Microscopy, Electron , Pituitary Gland, Anterior/ultrastructure , Rats , Rats, Inbred StrainsABSTRACT
The distribution of three components of basement membranes (BM): heparan-sulfate proteoglycan (HSPG), entactin (ENT), and type IV collagen (Coll. IV), was studied in the adult rat anterior pituitary and compared to the distribution of laminin (LAM) described in an earlier report (Vila-Porcile et al., 1987, J. Histochem. Cytochem., 35, 287). Several immunocytochemical methods were applied at both light and electron microscope levels. The three components were detected in all the pituitary BM and in endothelial and perivascular connective cells, as previously observed for LAM. In contrast to the prominent labeling previously observed for LAM within epithelial cells, however, only a faint immunoreaction could be detected for the other components, with nonetheless a discrete signal for Coll. IV. These findings indicate that 1) the four studied components are present in all the BM of the rat anterior pituitary; 2) pituitary endocrine cells contain LAM, and to a lesser extent Coll. IV, and thus could participate to the elaboration of the BM; and 3) the presence of the four components in non-epithelial cells also suggests their cooperation in BM building. The questions of the turnover and intracellular pathways of each of these components were addressed but remain unanswered.