Cell Proliferation, Chondrogenic Differentiation, and Cartilaginous Tissue Formation in Recombinant Silk Fibroin with Basic Fibroblast Growth Factor Binding Peptide.

Regeneration of articular cartilage remains a challenge for patients who have undergone cartilage injury, osteochondritis dissecans and osteoarthritis. Here, we describe a new recombinant silk fibroin with basic fibroblast growth factor (bFGF) binding peptide, which has a genetically introduced sequence PLLQATLGGGS, named P7. In this study, we cultured a human mesenchymal cell line derived from bone marrow, UE6E7-16, in wild-type fibroin sponge (FS) and recombinant silk fibroin sponge with P7 peptide (P7 FS). We compared cell proliferation, chondrogenic differentiation and cartilaginous tissue formation between the two types of sponge. After stimulation with bFGF at 3 ng/mL, P7 FS showed significantly higher cell growth (1.2-fold) and higher cellular DNA content (5.6-fold) than did wild-type FS. To promote chondrogenic differentiation, cells were cultured in the presence of TGF-ß at 10 ng/mL for 28 days. Immunostaining of P7 FS showed SOX9-positive cells comparable to wild-type FS. Alcian-Blue staining of P7 FS also showed cartilaginous tissue formation equivalent to wild-type FS. A significant increase in cell proliferation in P7 FS implies future clinical application of this transgenic fibroin for regeneration of articular cartilage. To produce cartilaginous tissue efficiently, transgenic fibroin sponges and culture conditions must be improved. Such changes should include the selection of growth factors involved in chondrogenic differentiation and cartilage formation.

Protein phosphatase 1H, overexpressed in colon adenocarcinoma, is associated with CSE1L.

In search for a new anticancer drug target, we explored genes involved in colon adenocarcinoma development through dysregulation of a signal transduction pathway. By using the gene expression profile database, we found protein phosphatase 1H (PPM1H), belonging to the protein phosphatase 2C (PP2C) family, upregulated in colon adenocarcinomas compared with normal colon tissues. RT-PCR analysis verified the elevated level of PPM1H expression in colon cancer cell lines relative to a normal colon cell line. PPM1H encodes a protein with a molecular mass of approximately 50 kDa that resides in the cytoplasm. PPM1H fused with maltose-binding protein expressed in E. coli exhibited phosphatase activity characteristic of the PP2C family. Co-immunoprecipitation coupled with mass spectrometry analysis identified CSE1L, a proliferation and apoptosis-related protein, as a PPM1H-interacting protein. Native, but not inactive, PPM1H expressed in HeLa cells increased the mobility of CSE1L on SDS gels and a similar mobility shift was observed for purified CSE1L after treatment with PPM1H in vitro, supporting the notion that CSE1L is a substrate of PPM1H. Dominant negative PPM1H protected HeLa cells from cell death triggered by staurosporine or taxol. Additionally, knockdown of PPM1H expression with small interfering RNAs suppressed the growth of MCF-7 cells weakly but consistently. PPM1H controls cell cycle and proliferation of cancer cells potentially through dephosphorylation of CSE1L and might be a new target of anticancer drugs.

Intracellular characterization of DDX39, a novel growth-associated RNA helicase.

DDX39 belongs to the DEAD box RNA helicase family and is overexpressed in human lung squamous cell carcinoma. In this study, in order to seek the biological relevance of DDX39, we conducted its intracellular characterization. When expressed in 293 cells, DDX39 undergoes heavy ubiquitylation and the stability of DDX39 is regulated via a ubiqutin-proteasome pathway. DDX39 tethers ALY, an essential mRNA export factor, in vivo, confirming the role of DDX39 in the RNA splicing/export process. Co-immunoprecipitation and mass spectrometry analyses detected CIP29, a recently discovered growth and cell cycle-related factor, as a main DDX39-interacting protein. CIP29 binds RNA on its own and enhances RNA unwinding activity of DDX39. Thus, CIP29 physically and functionally associates with DDX39, suggesting their cooperation in the RNA metabolism. Extension of the search for the protein-protein interactions encompassing DDX39 identified FUS/TLS, a nucleic acid binding protein participating in both transcription and splicing, as a CIP29-interacting protein. The connections comprising ALY, DDX39, CIP29 and FUS/TLS may be an integral part of transcription, splicing and RNA export. We simultaneously examined the properties of DDX39-S, a C-terminally truncated variant of DDX39 stemmed from alternative splicing, to understand its biological significance.