RESUMO
Streptococcus pyogenes is an important human pathogen that causes a variety of diseases including life-threatening invasive diseases, such as toxic shock and deep tissue infections. Although S. pyogenes are classically considered extracellular pathogens, a clinical significance of an intracellular source has been emphasized. In patients with deep tissue infections, an intracellular reservoir of S. pyogenes within macrophages was shown to contribute to prolonged bacterial persistence. Here we demonstrate that intracellular survival of S. pyogenes in macrophages is associated with an M1 protein-dependent intracellular trafficking in the phagosomal-lysosomal pathway, which results in impaired fusion with lysosomes. The phagocytic vacuoles harbouring M1 protein-expressing bacteria not only served as a safe haven for the bacteria, but also as a replicating niche. An M1 protein-dependent modulation of macrophages was further supported by differences in NF-κB signalling between cells infected with either the wild-type or M1 protein-deficient strains, thereby indicating a suppressed inflammatory response when M1 protein was involved. Evidence of egress of bacteria out of their host cell and subsequent re-infection of new cells emphasize the importance of intracellular bacteria as a reservoir for dissemination of infection and continued tissue injury.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/metabolismo , DNA Bacteriano/biossíntese , Macrófagos/metabolismo , Proteínas Mutantes/metabolismo , Infecções Estreptocócicas/imunologia , Streptococcus pyogenes/fisiologia , Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Transporte/genética , Células Cultivadas , Reservatórios de Doenças/microbiologia , Humanos , Evasão da Resposta Imune , Macrófagos/imunologia , Macrófagos/microbiologia , Macrófagos/patologia , Fusão de Membrana/genética , Proteínas Mutantes/genética , NF-kappa B/genética , NF-kappa B/metabolismo , Fagocitose/genética , Transporte Proteico/genética , Transdução de Sinais/imunologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/patogenicidadeRESUMO
During the process of differentiation, osteoblasts commit through strictly controlled checkpoints under the influence of several growth factors, cytokines, and extracellular matrix (ECM) proteins. The mineralized tissue-specific ECM component osteoadherin (OSAD) belongs to the small leucine-rich repeat protein family of proteoglycans. Proteoglycans modulate cellular behavior either through the attached glycosaminoglycan chains or by direct protein-protein interactions via the core protein sequences. Leucine-rich repeats have been shown to directly interact with cell-surface receptors such as epidermal growth factor receptor, blocking its ability to bind its ligand. In the present study, we investigated the influence of OSAD on the behavior and maturation of MC3T3E1 osteoblasts. OSAD overexpression and repression clones were created by stably transfecting with plasmids coding for either mouse OSAD cDNA or small-hairpin RNA, targeted against mouse OSAD. Overexpression of OSAD resulted in an increase of osteoblast differentiation features, such as increased alkaline phosphatase (ALP) activity and increased in vitro mineralization, as well as reduced proliferation and migration. Bone sialoprotein (BSP) levels were unchanged, while upregulation of osteocalcin (OC) and osteoglycin (OGN) was observed. Conversely, repression of OSAD expression resulted in increased cell proliferation and migration. BSP and OC were unaffected, while OGN was downregulated. ALP activity was reduced, though no change in in vitro mineralization was observed. We conclude that OSAD overexpression enhanced the differentiation and maturation of osteoblasts in vitro.
Assuntos
Calcificação Fisiológica/fisiologia , Proteínas da Matriz Extracelular/metabolismo , Osteoblastos/metabolismo , Proteoglicanas/metabolismo , Animais , Biomarcadores/metabolismo , Diferenciação Celular/fisiologia , Proteínas da Matriz Extracelular/genética , Expressão Gênica , Inativação Gênica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Proteoglicanas/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Regulação para CimaRESUMO
The multi-domain neutral endopeptidase, ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin repeats) is induced by parathyroid hormone (PTH) in rat osteoblasts and has therefore been suggested to be involved in initiation of bone remodeling. However, its function(s) in bone cells have not been studied. Here, we first establish that ADAMTS-1 protein is rapidly and transiently produced by human primary osteoblasts in response to PTH (1-34). We also show that ADAMTS-1 is specifically in close proximity to collagen fibrils in bone tissue using ultrastructural immunolabeling. To study the consequence(s) of ADAMTS-1 metalloprotease production in osteoblastic cells, human osteosarcoma cells (SaOS-2), were forced to express either wild-type (wtATS) or a point-mutated (pmATS) metalloprotease dead ADAMTS-1. SaOS-2 cells expressing wtATS had a growth advantage and increased collagenolytic activity when seeded inside a collagen type I gel but exhibited a reduced migration in a scratch wound assay. Immunolabeling of moving cells shows ADAMTS-1 to be located towards the direction of cellular migration. Finally, Western analysis demonstrated excess accumulation of mature collagen type I alpha1 species in the extracellular matrix together with increased release of distinct small collagen fragments into the conditioned media, by cultures of wtATS cells compared to pmATS cells. These results show that ADAMTS-1 has both the opportunity in bone and capability in vitro to induce collagen type I processing, together with a positive influence on osteoblastic three-dimensional growth. Although it is not clear at present if ADAMTS-1 promotes collagen degradation directly or indirectly, it shows that ADAMTS-1 activity can have a profound influence on the osteoblast phenotype, inhibiting migration on a planar substrate but enhancing growth in a collagen scaffold. These findings further establish ADAMTS-1 as a potentially important protein in PTH induced bone remodeling.
Assuntos
Proteínas ADAM/metabolismo , Colágeno Tipo I/metabolismo , Osteoblastos/fisiologia , Proteínas ADAM/genética , Proteína ADAMTS1 , Animais , Movimento Celular/fisiologia , Proliferação de Células , Células Cultivadas , Colágeno Tipo I/ultraestrutura , Humanos , Microscopia Imunoeletrônica , Osteoblastos/ultraestrutura , Osteossarcoma , Hormônio Paratireóideo/metabolismo , Ratos , Técnicas de Cultura de TecidosRESUMO
Osteoadherin (OSAD) is a member of the small leucine rich-repeat proteoglycan (SLRP) family. SLRPs are normally found in extracellular matrices, but OSAD is the only member restricted to mineralized tissues. We investigated the promoter region of OSAD by in silico analysis and found that the proximal promoter region contains sites for Smad-3, Smad-4, and AP-1. All are effectors of TGF-beta family signalling. We tested sensitivity of the promoter to the two TGF-beta family members TGF-beta1 and BMP-2. We found TGF-beta1 to down regulate OSAD, while BMP-2 up regulates OSAD. As a consequence of how OSAD is regulated by TGF-beta1 and BMP-2 and its temporal expression pattern in osteoblasts and bone development, we can conclude OSAD as an early marker for terminally differentiated matrix producing osteoblasts.
