Faculty Bibliography

p202, an interferon-inducible protein that belongs to an interferon-inducible p200 family, was highly induced in the course of osteogenesis of pluripotent C2C12 cells and the chondrogenesis of C3H10T1/2 cells. Differential expression of p202 is probably due, at least in part, to the transactivation of the p202 gene by Smad transcription factors. Overexpressing p202 inhibited, whereas lowering p202 via a siRNA approach enhanced, chondrocyte differentiation. In contrast, overexpression of p202 enhanced, whereas knockdown of p202 inhibited, osteoblast differentiation. Molecular mechanism studies revealed that p202 and parathyroid hormone-related peptide (PTHrP) formed a positive feedback loop, since (1) overexpressing p202 markedly enhanced whereas knocking down p202 suppressed the expression of PTHrP; and (2) p202 expression was increased in growth plate chondrocytes of PTHrP receptor transgenic mouse embryos; however, its expression was reduced in PTHrP knockout mouse embryos. Taken together, our findings demonstrate that p202 protein is a novel, important mediator of chondrogenic and osteogenic differentiation

The interferon-inducible p200 family comprises a group of homologous mouse and human proteins. Most of these have an N-terminal DAPIN domain and one or two partially conserved, 200 amino acid long C-terminal domains (designated as 200X domain). These proteins play important roles in the regulation of cell proliferation, tissue differentiation, apoptosis and senescence. p200 family proteins are involved also in autoimmunity and the control of tumor growth. These proteins function by binding to various target proteins (e.g. transcription factors, signaling proteins, oncoproteins and tumor suppressor proteins) and modulating target activity. This review concentrates on p204, a murine member of the family and its roles in regulating cell proliferation, cell and tissue differentiation (e.g. of skeletal muscle myotubes, beating cardiac myocytes, osteoblasts, chondrocytes and macrophages) and signaling by Ras proteins. The expression of p204 in various tissues as promoted by tissue-specific transcription factors, its distribution among subcellular compartments, and the controls of these features are also discussed

The authors report on a case of a patient who received recombinant human bone morphogenetic protein-2 (rhBMP-2) to augment spinal fusion for the first and third of 3 lumbosacral fusion surgeries. After receiving rhBMP-2 the first time, the patient became febrile and developed mild acute renal insufficiency and transient supraventricular tachycardia (SVT). The second operation was complicated only by perioperative fever. When the patient received rhBMP-2 again during the third operation, he developed fever, acute oliguric renal insufficiency, symptomatic SVT with hypoxemia, confusion, and joint pain. No clear cause of these problems was identified; however serum analysis revealed the presence of an antibody to rhBMP-2. The authors discuss potential mechanisms for the patient's putative reaction to rhBMP-2, as the findings from a literature review suggest this is the first such reaction to be reported

Id proteins play important roles in osteogenic differentiation; however, the molecular mechanism remains unknown. In this study, we established that inhibitor of differentiation (Id) proteins, including Id1, Id2, and Id3, associate with core binding factor alpha-1 (Cbfa1) to cause diminished transcription of the alkaline phosphatase (ALP) and osteocalcin (OCL) gene, leading to less ALP activity and osteocalcin (OCL) production. Id acts by inhibiting the sequence-specific binding of Cbfa1 to DNA and by decreasing the expression of Cbfa1 in cells undergoing osteogenic differentiation. p204, an interferon-inducible protein that interacts with both Cbfa1 and Id2, overcame the Id2-mediated inhibition of Cbfa1-induced ALP activity and OCL production. We show that 1) p204 disturbed the binding of Id2 to Cbfa1 and enabled Cbfa1 to bind to the promoters of its target genes and 2) that p204 promoted the translocation from nucleus to the cytoplasm and accelerated the degradation of Id2 by ubiquitin-proteasome pathway during osteogenesis. Nucleus export signal (NES) of p204 is required for the p204-enhanced cytoplasmic translocation and degradation of Id2, because a p204 mutant lacking NES lost these activities. Together, Cbfa1, p204, and Id proteins form a regulatory circuit and act in concert to regulate osteoblast differentiation

Although human papillomavirus (HPV) infections are the primary cause of cervical cancer, the molecular mechanism by which HPV induces cervical cancer remains largely unclear. We used two-dimensional electrophoresis with mass spectrometry to study protein expression profiling between HPV16-positive cervical mucosa epithelial H8 cells and cervical cancer Caski cells to identify 18 differentially expressed proteins. Among them, retinoblastoma-binding protein 4 (RbAp48) was selected and its differentiation expression verified with both additional cervical cancer-derived cell lines and human tissues of cervical intraepithelial neoplasia and cervical cancer. Suppression of RbAp48 using siRNA approach in H8 cells significantly stimulated cell proliferation and colony formation and inhibited senescence-like phenotype. Remarkably, H8 cells acquired transforming activity if RpAp48 was suppressed, since H8 cells stably transfected with RbAp48 siRNA led to tumor formation in nude mice. In addition, overexpression of RbAp48 significantly inhibited cell growth and tumor formation. This RbAp48-mediated transformation of HPV16 is probably due to the regulation by RbAp48 of tumor suppressors Rb and p53, apoptosis-related enzymes caspase-3 and caspase-8, and oncogenic genes, including cyclin D1 and c-myc. In brief, RbAp48, previously unknown in cervical carcinogenesis, was isolated in a global screen and identified as a critical mediator controlling the transforming activity of HPV16 in cervical cancer

IFI16 is a member of the interferon-inducible p200-protein family, capable of modulating cell proliferation, and cellular senescence. In this study, these effects of IFI16 were studied in tumor cells derived from bone and cartilage. The level of IFI16 was markedly lower in human osteosarcomas as compared with its level in normal bone. Overexpression of functional IFI16 in human osteosarcoma and chondrosarcoma cell lines markedly inhibited colony formation, and significantly inhibited cell growth, an effect that could be reversed by introduction of gene specific siRNA into tumor cells. These inhibitory effects of IFI16 were associated with upregulation of p21 and inhibition of cyclin E, cyclin D1, c-Myc and Ras. In addition, ectopic expression of IFI16 in tumor cells increased senescence-associated beta-galactosidase and induced a senescence-like phenotype. In view of such effects, IFI16 might be a suitable target for therapeutic intervention in osteosarcoma and chondrosarcoma.

Bone formation requires the coordinated activity of numerous proteins including the transcription factor core-binding factor alpha1 (Cbfa1). Deregulation of Cbfa1 results in metabolic bone diseases including osteoporosis and osteopetrosis. The retinoblastoma protein (pRb) that is required for osteogenesis binds Cbfa1. We reported earlier that the p200 family protein p204, which is known to be involved in the differentiation of skeletal muscle myotubes, cardiac myocytes, and macrophages, also serves as a cofactor of Cbfa1 and promotes osteogenesis. In this study we established that suppression of p204 expression by an adenovirus construct encoding p204 antisense RNA inhibited osteoblast-specific gene activation by Cbfa1 in an osteogenesis assay involving the pluripotent C2C12 mesenchymal cell line. Using protein-protein interaction assays we established that Cbfa1, pRb, and p204 form a ternary complex in which pRb serves as a linker connecting p204 and Cbfa1. Chromatin immunoprecipitation assays revealed the binding of such a p204-pRb-Cbfa1 transcription factor complex to the promoter of the osteocalcin gene. The pRb requirement of the stimulation of Cbfa1 activity by p204 was established in experiments involving p204 mutants lacking one or two pRb binding (LXCXE) motifs. Such mutants failed to enhance the Cbfa1-dependent transactivation of gene expression as well as osteogenesis. Furthermore, as revealed in reporter gene and in vitro osteogenesis assays p204 synergized with pRb in the stimulation of Cbfa1-dependent gene activation and osteoblast differentiation.

The gene for cartilage oligomeric matrix protein (COMP) encodes a noncollagenous matrix protein that is expressed predominantly in cartilage. COMP gene expression is deficient in the Sox9-null mouse, but the molecular mechanism remains unknown. We have previously delineated a 30-bp negative regulatory element (NRE) and a 51-bp positive regulatory element (PRE) in the regulatory region of the COMP gene. Subsequently we isolated LRF transcription repressor as an NRE-binding protein and established that LRF inhibits COMP gene expression via recruiting histone deacetylase 1 (HDAC1) to the COMP promoter. In this study we demonstrated that Sox9, an essential transcription factor of chondrogenesis, binds to the COMP promoter at the PRE in which 13 nucleotides (TGTTTACCTTGTG) are required for the binding of Sox9. Sox9 activates COMP gene expression and this activation is PRE-dependent. Sox9 is required for COMP gene expression during chondrogenesis, since repression of Sox9 expression using the small interfering RNA approach inhibited COMP gene expression. In addition, activation of COMP gene expression by Sox9 requires the participation of transcription factors Sox5 and Sox6 as well as the coactivators CBP and p300 histone acetylase. It appears that there exists a balance between LRF repressor and Sox9 activator in the control of COMP gene, since transactivation of COMP gene by Sox9 was abolished by the coexpression of LRF, and excess Sox9 overcame the LRF-mediated inhibition. This study provides the first evidence that Sox9 directly associates with COMP gene promoter and that mediation of COMP gene activation by Sox9 involves Sox5, Sox6, CBP, and p300 coactivators.