RESUMO
Podocytes play a major role in the initiation and progression of glomerular diseases and are a target of both immune-mediated and non-immune-mediated injury. To establish a mouse model of such injury, we preimmunized mice with Freunds adjuvant 5 days before intravenous injection of a rabbit polyclonal antibody directed against a murine podocyte cell line. For the next 7 weeks, we collected urine, serum, and kidney samples. Nephritic animals developed severe albuminuria, which was maximal on day 10. Histochemistry revealed diffuse mesangial matrix expansion. Mouse immunoglobulin G and complement were detected in a linear pattern along the glomerular filtration barrier and in the mesangial hinge region. Complement depletion, however, did not prevent proteinuria. Glomerular T cells were increased, whereas podocytes were significantly reduced. Glomerular foot processes were flattened in regions with mesangial matrix deposition as viewed by electron microscopy. Immunohistochemistry detected the injected anti-podocyte antibody exclusively at the glomerular tuft on all days examined. Immunoelectron microscopy localized the antibody to podocyte foot processes and the glomerular basement membrane, which was morphologically intact. This suggests that the podocyte was the main target of the antiserum. Our study establishes a new mouse model of immune-mediated podocyte injury.
Assuntos
Modelos Animais de Doenças , Glomerulonefrite/patologia , Soros Imunes , Podócitos/patologia , Proteinúria , Animais , Anticorpos/metabolismo , Nitrogênio da Ureia Sanguínea , Complemento C3/metabolismo , Fibrose , Glomerulonefrite/imunologia , Glomerulonefrite/metabolismo , Imunoglobulina G/metabolismo , Imunoglobulina M/metabolismo , Imuno-Histoquímica , Glomérulos Renais/imunologia , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Contagem de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Imunoeletrônica , Podócitos/metabolismo , Coelhos , EscleroseRESUMO
DNA polymerase alpha-primase (pol-prim) is the only enzyme that can start DNA replication de novo. The 180-kDa (p180) and 68-kDa (p68) subunits of the human four-subunit enzyme are phosphorylated by Cyclin-dependent kinases (Cdks) in a cell cycle-dependent manner. Cyclin A-Cdk2 physically interacts with pol-prim and phosphorylates N-terminal amino acids of the p180 and the p68 subunits, leading to an inhibition of pol-prim in initiating cell-free SV40 DNA replication. Mutation of conserved putative Cdk phosphorylation sites in the N terminus of human p180 and p68 reduced their phosphorylation by Cyclin A-Cdk2 in vitro. In contrast to wild-type pol-prim these mutants were no longer inhibited by Cyclin A-Cdk2 in the initiation of viral DNA replication. Importantly, rather than inhibiting it, Cyclin A-Cdk2 stimulated the initiation activity of pol-prim containing a triple N-terminal alanine mutant of the p180 subunit. Together these results suggest that Cyclin A-Cdk2 executes both stimulatory and inhibitory effects on the activity of pol-prim in initiating DNA replication.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , DNA Polimerase I/metabolismo , DNA Primase/metabolismo , Replicação do DNA , Animais , Células Cultivadas , Ciclina A/metabolismo , Quinase 2 Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , DNA Polimerase I/química , DNA Primase/química , Humanos , Insetos , Fosforilação , Testes de Precipitina , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Metabolic labeling of primate cells revealed the existence of phosphorylated and hypophosphorylated DNA polymerase alpha-primase (Pol-Prim) populations that are distinguishable by monoclonal antibodies. Cell cycle studies showed that the hypophosphorylated form was found in a complex with PP2A and cyclin E-Cdk2 in G1, whereas the phosphorylated enzyme was associated with a cyclin A kinase in S and G2. Modification of Pol-Prim by PP2A and Cdks regulated the interaction with the simian virus 40 origin-binding protein large T antigen and thus initiation of DNA replication. Confocal microscopy demonstrated nuclear colocalization of hypophosphorylated Pol-Prim with MCM2 in S phase nuclei, but its presence preceded 5-bromo-2'-deoxyuridine (BrdU) incorporation. The phosphorylated replicase exclusively colocalized with the BrdU signal, but not with MCM2. Immunoprecipitation experiments proved that only hypophosphorylated Pol-Prim associated with MCM2. The data indicate that the hypophosphorylated enzyme initiates DNA replication at origins, and the phosphorylated form synthesizes the primers for the lagging strand of the replication fork.
Assuntos
DNA Primase/genética , Replicação do DNA , Animais , Anticorpos Monoclonais/imunologia , Linhagem Celular , DNA Primase/imunologia , Humanos , Isoenzimas/genética , Isoenzimas/imunologia , Microscopia Confocal , FosforilaçãoRESUMO
Microinjection of purified simian virus 40 large-T-antigen protein or DNA encoding T antigen into serum-starved cells stimulates them to re-enter the cell cycle and progress through G1 into the S phase. Genetic analysis of T antigen indicated that neither its Rb/p107-binding activity nor its p53-binding activity is essential to induce DNA synthesis in CV1P cells. However, T antigens bearing missense mutations that inactivate either activity induced slower progression of the cells into the S phase than did wild-type T antigen. Inactivation of both activities resulted in a T antigen essentially unable to induce DNA synthesis. Missense mutations in either the DNA-binding region of the N terminus also impaired the ability of full-length T antigen to stimulate DNA synthesis in CV1P cells. The wild-type kinetics of cell cycle progression were restored by genetic complementation after coinjection of plasmid DNAs encoding different mutant T antigens or coinjection of purified mutant T-antigen proteins, suggesting that the four mitogenic functions of T antigen are independent. The maximal rate of induction of DNA synthesis in secondary primate cells and established rodent cell lines required the same four functions of T antigen. A model to explain how four independent activities could cooperate to stimulate cell cycle progression is presented.
Assuntos
Antígenos Transformantes de Poliomavirus/fisiologia , Ciclo Celular , Regulação Viral da Expressão Gênica , Vírus 40 dos Símios/genética , Animais , Linhagem Celular , Teste de Complementação Genética , Haplorrinos , RNA Mensageiro/genética , Proteínas Recombinantes , Proteína do Retinoblastoma/fisiologia , Fatores de Tempo , Proteína Supressora de Tumor p53/fisiologiaRESUMO
The ability of the oncogene products of DNA tumor viruses to induce DNA synthesis in quiescent cells is thought to depend on their capacity to bind to cellular proteins such as the retinoblastoma-suppressor protein Rb and the tumor suppressor p53, thereby abolishing the growth-arresting properties of these proteins. We have tested this hypothesis using SV40 T antigens carrying lesions that affect Rb binding, p53 binding or other functions involved in cell transformation. The results demonstrate that Rb binding is not essential for growth stimulation by T antigen. However, detailed analysis, including intracistronic complementation, suggests that at least three functions, Rb binding, a novel second activity localized to the DNA-binding domain and a function residing in the carboxy terminus, probably p53 binding, cooperate to generate the full growth induction potential of T antigen.