Shuji Muramatsu

Large-scale identification and characterization of human genes that activate NF-kappaB and MAPK signaling pathways.

We have carried out a large-scale identification and characterization of human genes that activate the NF-κB and MARK signaling pathways. We constructed full-length cDNA libraries using the oligo-capping method and prepared an arrayed cDNA pool consisting of 150 000 cDNAs randomly isolated from the libraries. For analysis of the NF-κB signaling pathway, we introduced each of the cDNAs into human embryonic kidney 293 cells and examined whether it activated the transcription of a luciferase reporter gene driven by a promoter containing the consensus NF-κB binding sites. In total, we identified 299 cDNAs that activate the NF-κB pathway, and we classified them into 83 genes, including 30 characterized activator genes of the NF-κB pathway, 28 genes whose involvement in the NF-κB pathways have not been characterized and 25 novel genes. We then carried out a similar analysis for the identification of genes that activate the MARK pathway, utilizing the same cDNA resource. We assayed 145 000 cDNAs and identified 57 genes that activate the MARK pathway. Interestingly, 27 genes were overlapping between the NF-κB and the MAPK pathways, which may indicate that these genes play cross-talking roles between these two pathways.

Functional gene screening system identified TRPV4 as a regulator of chondrogenic differentiation.

Sox9 is a transcription factor that is essential for chondrocyte differentiation and chondrocyte-specific gene expression. However, the precise mechanism of Sox9 activation during chondrogenesis is not fully understood. To investigate this mechanism, we performed functional gene screening to identify genes that activate SOX9-dependent transcription, using full-length cDNA libraries generated from a murine chondrogenic cell line, ATDC5. Screening revealed that TRPV4 (transient receptor potential vanilloid 4), a cation channel molecule, significantly elevates SOX9-dependent reporter activity. Microarray and quantitative real time PCR analyses demonstrated that during chondrogenesis in ATDC5 and C3H10T1/2 (a murine mesenchymal stem cell line), the expression pattern of TRPV4 was similar to the expression patterns of chondrogenic marker genes, such as type II collagen and aggrecan. Activation of TRPV4 by a pharmacological activator induced SOX9-dependent reporter activity, and this effect was abolished by the addition of the TRPV antagonist ruthenium red or by using a small interfering RNA for TRPV4. The SOX9-dependent reporter activity due to TRPV4 activation was abrogated by both EGTA and a calmodulin inhibitor, suggesting that the Ca2+/calmodulin signal is essential in this process. Furthermore, activation of TRPV4 in concert with insulin activity in ATDC5 cells or in concert with bone morphogenetic protein-2 in C3H10T1/2 cells promoted synthesis of sulfated glycosaminoglycan, but activation of TRPV4 had no effect alone. We showed that activation of TRPV4 increased the steady-state levels of SOX9 mRNA and protein and SOX6 mRNA. Taken together, our results suggest that TRPV4 regulates the SOX9 pathway and contributes to the process of chondrogenesis.

Paraspeckle protein p54nrb links Sox9-mediated transcription with RNA processing during chondrogenesis in mice

The Sox9 transcription factor plays an essential role in promoting chondrogenesis and regulating expression of chondrocyte extracellular-matrix genes. To identify genes that interact with Sox9 in promoting chondrocyte differentiation, we screened a cDNA library generated from the murine chondrogenic ATDC5 cell line to identify activators of the collagen, type II, alpha 1 (Col2a1) promoter. Here we have shown that paraspeckle regulatory protein 54-kDa nuclear RNA-binding protein (p54nrb) is an essential link between Sox9-regulated transcription and maturation of Sox9-target gene mRNA. We found that p54nrb physically interacted with Sox9 and enhanced Sox9-dependent transcriptional activation of the Col2a1 promoter. In ATDC5 cells, p54nrb colocalized with Sox9 protein in nuclear paraspeckle bodies, and knockdown of p54(nrb) suppressed Sox9-dependent Col2a1 expression and promoter activity. We generated a p54nrb mutant construct lacking RNA recognition motifs, and overexpression of mutant p54nrb in ATDC5 cells markedly altered the appearance of paraspeckle bodies and inhibited the maturation of Col2a1 mRNA. The mutant p54nrb inhibited chondrocyte differentiation of mesenchymal cells and mouse metatarsal explants. Furthermore, transgenic mice expressing the mutant p54nrb in the chondrocyte lineage exhibited dwarfism associated with impairment of chondrogenesis. These data suggest that p54nrb plays an important role in the regulation of Sox9 function and the formation of paraspeckle bodies during chondrogenesis.

The transcription factor Znf219 regulates chondrocyte differentiation by assembling a transcription factory with Sox9

Sox9 is an essential transcription factor for chondrogenesis by regulating the expression of chondrogenic genes. However, its regulatory mechanism is not fully understood. To address this, we attempted to identify the transcriptional partners of Sox9 by screening the cDNA library of the chondrogenic cell line ATDC5 using the collagen 2α1 (Col2α1) gene promoter fused to a luciferase reporter gene. One of the positive clones encoded the Znf219 gene. Whole mount in situ hybridization experiments indicated that Znf219 mRNA was specifically expressed in the developing limb buds where Col2α1 and Sox9 were strongly expressed. Znf219 markedly enhanced the transcriptional activity of Sox9 on the Col2a1 gene promoter. In addition, Znf219 is physically associated with Sox9 and is colocalized with Sox9 in the nucleus. We also found that overexpression of Znf219 profoundly increased Sox9-induced mRNA expression of Col2a1, aggrecan and Col11a2. Consistently, knockdown of Znf219 decreased the Sox9-induced mRNA expression of these genes. Furthermore, a dominant-negative mutant Znf219 inhibited Bmp2-induced chondrocyte differentiation. Our results suggest that Znf219 plays an important role in the regulation of chondrocyte differentiation as a transcriptional partner of Sox9.

Centaurin-α1 Is a Phosphatidylinositol 3-Kinase-dependent Activator of ERK1/2 Mitogen-activated Protein Kinases

Centaurin-α1 is known to be a phosphatidylinositol 3,4,5-triphosphate (PIP3)-binding protein that has two pleckstrin homology domains and a putative ADP ribosylation factor GTPase-activating protein domain. However, the physiological function of centaurin-α1 is still not understood. Here we have shown that transient expression of centaurin-α1 in COS-7 cells results in specific activation of ERK, and the activation is inhibited by co-expression of a dominant negative form of Ras. We have also found that a mutant form of centaurin-α1 that is unable to bind PIP3 fails to induce ERK activation and that a phosphatidylinositol 3-kinase inhibitor LY294002 inhibits centaurin-α1-dependent ERK activation. Furthermore, transient knockdown of centaurin-α1 by small interfering RNAs results in reduced ERK activation after epidermal growth factor stimulation in T-REx 293 cells. These results suggest that centaurin-α1 contributes to ERK activation in growth factor signaling, linking the PI3K pathway to the ERK mitogen-activated protein kinase pathway through its ability to interact with PIP3.

Arid5a cooperates with Sox9 to stimulate chondrocyte-specific transcription

This study shows that Arid5a interacts with Sox9 and subsequently modulates histone 3 acetylation of a chondrogenic gene, Col2a1, and stimulates chondrocyte differentiation.