Tetraspanins CD9 and CD81 facilitate the fusion between gametes, myoblasts, or virus-infected cells. Here, we investigated the role of these tetraspanins in the fusion of mononuclear phagocytes. Expression of CD9 and CD81 and their complex formation with integrins were up-regulated when blood monocytes were cultured under normal conditions. Under fusogenic conditions in the presence of Con A, CD9 and CD81 up-regulation was inhibited, and their complex formation with integrins was down-regulated. Anti-CD9 and -CD81 antibodies, which were previously shown to inhibit the fusion of gametes, myoblasts, and virus-infected cells, unexpectedly promoted the fusion of monocytes and alveolar macrophages. However, these effects were not due to altered cell adhesion, aggregation, or cytokine production. When stimulated in vitro or in vivo, alveolar macrophages and bone marrow cells of CD9- and CD81-null mice formed larger numbers of multinucleated cells than those of wild-type mice. Finally, CD9/CD81 double-null mice spontaneously developed multinucleated giant cells in the lung and showed enhanced osteoclastogenesis in the bone. These results suggest that CD9 and CD81 coordinately prevent the fusion of mononuclear phagocytes.

Lipocalin-type prostaglandin D synthase is responsible for the biosynthesis of prostaglandin D 2 in the central nervous system and the genital organs and is secreted into the cerebrospinal fluid and the seminal plasma as ␤-trace. Here we analyzed retinoids binding of the enzyme by monitoring the fluorescence quenching of an intrinsic tryptophan residue, and appearance of circular dichroism around 330 nm, and a red shift of the UV absorption spectra of retinoids. We found that the enzyme binds all-trans-or 9-cis-retinoic acid and alltrans-or 13-cis-retinal, but not all-trans-retinol, with affinities (K d of 70 -80 nM) sufficient for function as a retinoid transporter. All-trans-retinoic acid inhibited the enzyme activity in a noncompetitive manner, suggesting that it binds to the same hydrophobic pocket as prostaglandin H 2 , the substrate for prostaglandin D synthase, but at a different site in this pocket. It is likely that this enzyme is a bifunctional protein that acts as both retinoid transporter and prostaglandin D 2 -producing enzyme.Retinoids play an important role in regulating a variety of biological processes, including differentiation, morphogenesis, and cell proliferation. The process is initiated by retinoid binding to the nuclear receptor for retinoic acid (RAR/RXR) 1 ; RAR binds both all-trans-and 9-cis-retinoic acids, whereas RXR is specific for the 9-cis-isomer (1-3). The binding of retinoic acids to the dimerized receptor, RAR-RXR or RXR-RXR, activates or inhibits the transcription of retinoid-responsive genes. The proteins that transport retinoids are divided into two distinct families based on their sequence, structure, and function: the secretory transporters and the intracellular transporters (4). Secretory retinoid transporters, such as plasma retinol-binding protein (RBP) and ␤-lactoglobulin, circulate retinoids in a variety of body fluids and transport them to the intracellular retinoid transporters, cellular RBP (CRBP), and cellular retinoic acid-binding protein (CRABP), which finally transfer the retinoids to RAR or RXR.Retinoids also regulate a number of genes expressed in the central nervous system and thus play a variety of important roles, particularly in development (5, 6). In the brain, mRNAs for RAR ␤1 and ␤3 (5, 7) and for RXR ␤ and ␥ (5, 7) were found as well as CRBP and CRABP (8, 9). However, secretory retinoid transporters in the central nervous system have not been identified, although retinoids would need to change transporters at the blood-brain barrier as the barrier is impermeable to the secretory transporter.Recently, a major protein in human cerebrospinal fluid, classically termed ␤-trace (10), was identified as prostaglandin (PG) D synthase (11-14). The enzyme is responsible for biosynthesis of PGD 2 , which is a major PG in the brain of various mammals, including humans, and is proposed to be an endogenous sleep-promoting substance (15, 16), as well as a modulator of several central actions, such as the regulation of body temperature, luteinizing hormone release, an...

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Efficient differentiation of human pluripotent stem cells (hPSCs) into neurons is paramount for disease modeling, drug screening, and cell transplantation therapy in regenerative medicine. In this manuscript, we report the capability of five transcription factors (TFs) toward this aim: NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2. In contrast to previous methods that have shortcomings in their speed and efficiency, a cocktail of these TFs as synthetic mRNAs can differentiate hPSCs into neurons in 7 days, judged by calcium imaging and electrophysiology. They exhibit motor neuron phenotypes based on immunostaining. These results indicate the establishment of a novel method for rapid, efficient, and footprint-free differentiation of functional neurons from hPSCs.

Small cell lung cancer (SCLC) invades locally and metastasizes distantly extremely early when compared with nonsmall cell lung cancer (NSCLC). The underlying molecular mechanisms, however, have not been elucidated. Accumulating evidence suggests that downregulation of several members of tetraspanins is associated with progression of solid tumors, thus indicating poor prognosis. Here we screened 30 lung cancer cell lines for expression of tetraspanins, CD9, CD63, CD81, CD82, CD151, and NAG-2. Flow cytometry revealed that, among these proteins, CD9 is broadly expressed in NSCLC lines, but is absent or highly reduced in most SCLC lines (Po0.0001). Using the Boyden chamber and videomicroscopic cell motility assays, we showed that stable transfection of CD9 into an SCLC line, OS3-R5, reduced cell motility on fibronectin. Furthermore, by transient transfection of green fluorescent protein (GFP)-tagged CD9 into three other SCLC lines, we observed that SCLC cells expressing GFP-CD9 were uniformly less motile than untransfected cells. CD9 or GFP-CD9 was associated with b1 integrins and distributed at the tumor cell periphery and cell-cell contacts, suggesting that CD9 modifies b1 integrin function to reduce motility. These findings suggest that low expression of CD9 may contribute to the highly invasive and metastatic phenotype of SCLC.