RESUMO
OBJECTIVE: The platelet-derived growth factor (PDGF) family consists of four members, PDGF A, PDGF B, and 2 new members, PDGF C and PDGF D, which signal through the alpha and beta PDGF receptor (PDGFR) tyrosine kinases. This study was performed to determine the receptor specificity and cellular expression profile of PDGF C. METHODS AND RESULTS: PDGF C growth factor domain (GFD) was shown to preferentially bind and activate alpha PDGFR and activate beta PDGFR when it is co-expressed with alpha PDGFR through heterodimer formation. An investigation of PDGF C mRNA and protein expression revealed that during mouse fetal development, PDGF C was expressed in the mesonephric mesenchyme, prefusion skeletal muscle, cardiac myoblasts, and in visceral and vascular smooth muscle, whereas in adult human tissues expression was largely restricted to smooth muscle. Microarray analysis of various cell types showed PDGF C expression in vascular smooth muscle cells, renal mesangial cells, and platelets. PDGF C mRNA expression in platelets was confirmed by real-time polymerase chain reaction, and PDGF C protein was localized in alpha granules by immuno-gold electron microscopy. Western blot analysis of platelets identified 55-kDa and 80-kDa PDGF C isoforms that were secreted on platelet activation. CONCLUSIONS: Taken together, our results demonstrated for the first time to our knowledge that like PDGF A and B, PDGF C is likely to play a role in platelet biology.
Assuntos
Fator de Crescimento Derivado de Plaquetas/fisiologia , Animais , Plaquetas/metabolismo , Plaquetas/ultraestrutura , Linhagem Celular/metabolismo , Grânulos Citoplasmáticos/química , DNA Complementar/genética , Dimerização , Desenvolvimento Embrionário e Fetal , Endopeptidases/sangue , Humanos , Linfocinas , Camundongos/embriologia , Camundongos Endogâmicos BALB C , Músculo Liso Vascular/metabolismo , Especificidade de Órgãos , Fosforilação , Ativação Plaquetária , Fator de Crescimento Derivado de Plaquetas/química , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-sis/química , Proteínas Proto-Oncogênicas c-sis/metabolismo , RNA Mensageiro/biossíntese , Receptores do Fator de Crescimento Derivado de Plaquetas/química , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Recombinantes de Fusão/fisiologia , TransfecçãoRESUMO
Applications of microarray technologies to mouse embryology/genetics have been limited, due to the nonavailability of microarrays containing large numbers of embryonic genes and the gap between microgram quantities of RNA required by typical microarray methods and the miniscule amounts of tissue available to researchers. To overcome these problems, we have developed a microarray platform containing in situ-synthesized 60-mer oligonucleotide probes representing approximately 22,000 unique mouse transcripts, assembled primarily from sequences of stem cell and embryo cDNA libraries. We have optimized RNA labeling protocols and experimental designs to use as little as 2 ng total RNA reliably and reproducibly. At least 98% of the probes contained in the microarray correspond to clones in our publicly available collections, making cDNAs readily available for further experimentation on genes of interest. These characteristics, combined with the ability to profile very small samples, make this system a resource for stem cell and embryogenomics research.