Expression analysis of LacZ gene placed in the locus of Cnot7 exhibits its activity in osteoblasts in vivo and in mineralized nodules in vitro

CCR4-NOT complex 7 (Cnot7) was identified as a regulator of gene expression in yeast and evolutionally conserved in mammals. Cnot7 deficient male mice exhibit abnormality in spermatogenesis. As these mice contained construct to express LacZ, we followed the expression patterning in these animals. LacZ was expressed in osteoblasts located in the primary spongiosa in adult mice. Cellular analysis indicated that LacZ is expressed in osteoblasts but not in osteoclasts. In the mineralized nodules formed in the culture of bone marrow cells obtained from Cnot7 +/- mice, LacZ expression was mainly observed in the cells forming mineralized nodules but not in un-mineralized area scattered around the periphery of the nodules. LacZ blue positive cells were gradually depositing minerals along its time course of the in vitro mineralization assay. Cnot7 expression was enhanced by the treatment with BMP. These data suggest that Cnot7 is expressed in osteoblasts and is associated with mineralization.     

Activation of genes for growth factor and cytokine pathways late in chondrogenic differentiation of ATDC5 cells

The mouse embryonal carcinoma cell line ATDC5 provides an excellent model system for chondrogenesis in vitro. To understand better the molecular mechanisms of endochondral bone formation, we investigated gene expression profiles during the differentiation course of ATDC5 cells, using an in-house microarray harboring full-length-enriched cDNAs. For 28 days following chondrogenic induction, 507 genes were up- or down-regulated at least 1.5-fold. These genes were classified into five clusters based on their expression patterns. Genes for growth factor and cytokine pathways were significantly enriched in the cluster characterized by increases in expression during late stages of chondrocyte differentiation. mRNAs for decorin and osteoglycin, which have been shown to bind to transforming growth factors-beta and bone morphogenetic proteins, respectively, were found in this cluster and were detected in hypertrophic chondrocytes of developing mouse bones by in situ hybridization analysis. Taken together with assigned functions of individual genes in the cluster, interdigitated interaction between a number of intercellular signaling molecules is likely to take place in the late chondrogenic stage for autocrine and paracrine regulation among chondrocytes, as well as for chemoattraction and stimulation of progenitor cells of other lineages.     

Sphingosine-1-phosphate phosphohydrolase regulates endoplasmic reticulum-to-golgi trafficking of ceramide

Previous studies demonstrated that sphingosine-1-phosphate (S1P) phosphohydrolase 1 (SPP-1), which is located mainly in the endoplasmic reticulum (ER), regulates sphingolipid metabolism and apoptosis (H. Le Stunff et al., J. Cell Biol. 158:1039-1049, 2002). We show here that the treatment of SPP-1-overexpressing cells with S1P, but not with dihydro-S1P, increased all ceramide species, particularly the long-chain ceramides. This was not due to inhibition of ceramide metabolism to sphingomyelin or monohexosylceramides but rather to the inhibition of ER-to-Golgi trafficking, determined with the fluorescent ceramide analog N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-d-erythro-sphingosine (DMB-Cer). Fumonisin B1, an inhibitor of ceramide synthase, prevented S1P-induced elevation of all ceramide species and corrected the defect in ER transport of DMB-Cer, readily allowing its detection in the Golgi. In contrast, ceramide accumulation had no effect on either the trafficking or the metabolism of 6-([N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]hexanoyl)-sphingosine, which rapidly labels the Golgi even at 4 degrees C. Protein trafficking from the ER to the Golgi, determined with vesicular stomatitis virus ts045 G protein fused to green fluorescent protein, was also inhibited in SPP-1-overexpressing cells in the presence of S1P but not in the presence of dihydro-S1P. Our results suggest that SPP-1 regulates ceramide levels in the ER and thus influences the anterograde membrane transport of both ceramide and proteins from the ER to the Golgi apparatus.     

A eukaryotic capsular polysaccharide is synthesized intracellularly and secreted via exocytosis

Cryptococcus neoformans, which causes fatal infection in immunocompromised individuals, has an elaborate polysaccharide capsule surrounding its cell wall. The cryptococcal capsule is the major virulence factor of this fungal organism, but its biosynthetic pathways are virtually unknown. Extracellular polysaccharides of eukaryotes may be made at the cell membrane or within the secretory pathway. To test these possibilities for cryptococcal capsule synthesis, we generated a secretion mutant in C. neoformans by mutating a Sec4/Rab8 GTPase homolog. At a restrictive temperature, the mutant displayed reduced growth and protein secretion, and accumulated approximately 100-nm vesicles in a polarized manner. These vesicles were not endocytic, as shown by their continued accumulation in the absence of polymerized actin, and could be labeled with anti-capsular antibodies as visualized by immunoelectron microscopy. These results indicate that glucuronoxylomannan, the major cryptococcal capsule polysaccharide, is trafficked within post-Golgi secretory vesicles. This strongly supports the conclusion that cryptococcal capsule is synthesized intracellularly and secreted via exocytosis.     

Synthesis of polyamine derivatives having non-hypotensive Ca2 +-permeable AMPA receptor antagonist activity

In order to obtain non-hypotensive and Ca2+-permeable AMPA receptor antagonists, we have synthesized a series of 1,4-bis(4-piperidinylmethyl)diaminobutanes. Compounds 13b, c, f had desirable properties.     

Osteoclastogenesis is negatively regulated by D-serine produced by osteoblasts

We have shown the functional expression by chondrocytes of serine racemase (SR) which is responsible for the synthesis of D-serine (Ser) from L-Ser in cartilage. In this study, we evaluated the possible functional expression of SR by bone-forming osteoblasts and bone-resorbing osteoclasts. Expression of SR mRNA was seen in osteoblasts localized at the cancellous bone surface in neonatal rat tibial sections and in cultured rat calvarial osteoblasts endowed to release D-Ser into extracellular medium, but not in cultured osteoclasts differentiated from murine bone marrow progenitor cells. Sustained exposure to D-Ser failed to significantly affect alkaline phosphatase activity and Ca(2+) accumulation in cultured osteoblasts, but significantly inhibited differentiation and maturation in a concentration-dependent manner at a concentration range of 0.1-1 mM without affecting cellular survival in cultured osteoclasts. By contrast, L-Ser promoted osteoclastic differentiation in a manner sensitive to the inhibition by D-Ser. Matured osteoclasts expressed mRNA for the amino acid transporter B(0,+) (ATB(0,+) ) and the system alanine, serine, and cysteine amino acid transporter-2 (ASCT2), which are individually capable of similarly incorporating extracellular L- and D-Ser. Knockdown of these transporters by siRNA prevented both the promotion by L-Ser and the inhibition by D-Ser of osteoclastic differentiation in pre-osteoclastic RAW264.7 cells. These results suggest that D-Ser may play a pivotal role in osteoclastogenesis through a mechanism related to the incorporation mediated by both ATB(0,+) and ASCT2 of serine enantiomers in osteoclasts after the synthesis and subsequent release from adjacent osteoblasts.