Biological Studies On Interferon Inducibale Protein 200 Family

The interferon-inducible p200 (IFI-200) family proteins is among the numerous gene products induced by interferons (IFNs), which are important regulators of cell growth, immunomodulation and host resistance to tumors and viral infections. The members of this family of proteins are highly homologous to one another and consist of four murine proteins including p202, p203, p204 and D3 as well as three human homologues; IFI-16, myeloid cell nuclear differentiation antigen (MNDA) and absent in melanoma (AIM) 2. They possess at least one copy of a conserved 200 amino-acid motif which exists in two types; the a and b domains. Most of the IFI-200 proteins also possess a domain in apoptosis and interferon response (DAPIN)/PYRIN domain, which is a conserved motif associated with protein-protein interactions in the regulation of apoptotic and inflammatory signaling pathways. The p200 proteins have been implicated in cell cycle regulation and differentiation based on their ability to interact with and modulate the activities of multiple transcriptional factors such as Rb and p53, and there are significant findings that link mutations in their genetic loci to the incidence of cancer.My researches focus on p204, a murine member of IFI200 family and IFI16, the human counterpart of p204 in the same family.IFI204 gene was first cloned in Dr.Lengyel's lab at Yale University approximately a decade ago. It consists of 640 amino acid residues with an apparent molecular mass of 72 kDa. In the N-terminal domain there exist a basic amino acid-rich nuclear localization signal (NLS) and a canonical nuclear export signal (NES) required for the translocation of p204 from the nucleus to the cytoplasm during myoblast differentiation ; p204 consists of two conserved 200-amino-acid segments (both a and b). Over-expression of p204 is growth-inhibitory, probably due to its inhibition of rRNA transcription by binding to the ribosomal DNA-specific upstream binding factor (UBF) transcription factor and inhibiting its sequence-specific binding to DNA. p204 binds to pRb via itspRb-binding motifs LXCXE, and play an important role in cell's growth inhibition.p204 is expressed in many tissue and organs, such as thymus, bone, marrow, spleen, heart and skeletal muscle. Previous investigations from Dr. Lengyel's lab revealed that p204 plays important roles in myogenesis. Overexpress of p204 in pluriopotent C2C12 cell can induce the fusion of this myoblast into myotube. In the course of this processes, p204 overcome the inhibition of Ids on MyoD, an essential transcriptional factor for myogenesis. Little is known on the functions of p204 in the regulations of commitment and differentiation of other cell lineages. This study is to investigate the roles of p204 in osteoblast differentiation and osteogenesis. comprehensive assays using approaches in modern immunohistochemistry, biochemistry, molecular-biology and genetics clearly demoi .trated that p204 is also involved in the modulation of osteogenesis as well as its related molecular mechanism.Western Blotting assay clearly revealed that p204 is expressed in the osteoblasts of postnatal mice and immunostaining assays using tibial growth plates of mouse embryos on postcoital (p.c.) day 19 showed that p204 is also highly expressed in embryonic osteoblasts and predominantly localized to the nucleus. In the growth plate, p204 is highly expressed in differentiating hypertrophic chondrocytes but is absent in both resting and proliferating chondrocytes; these results suggest that p204 plays an important role in bone longitudinal growth. A 1.6-kb segment from the 5'-flanking region of the Ifi204 gene was found to contain at least five Smad-binding consensus sequences. This finding prompted us to test for the involvement of Smads-binding elements (SBEs) in the induction of p204 during osteoblast differentiation. Two reporter gene plasmids, 204-2SBE-luc and 204-5SBE-luc, were generated in which segments with SBEs from the 5'-flanking region of Ifi204 (-1578 to -1324 and -1578 to +38) were linked to the upstream end of a re...