The Role of Parathyroid Hormone-Related Protein (PTHrP) in Osteoblast Response to Microgravity: Mechanistic Implications for Osteoporosis Development.

Prolonged skeletal unloading through bedrest results in bone loss similar to that observed in elderly osteoporotic patients, but with an accelerated timeframe. This rapid effect on weight-bearing bones is also observed in astronauts who can lose up to 2% of their bone mass per month spent in Space. Despite the important implications for Spaceflight travelers and bedridden patients, the exact mechanisms involved in disuse osteoporosis have not been elucidated. Parathyroid hormone-related protein (PTHrP) regulates many physiological processes including skeletal development, and has been proposed as a mechanosensor. To investigate the role of PTHrP in microgravity-induced bone loss, trabecular and calvarial osteoblasts (TOs and COs) from Pthrp +/+ and -/- mice were subjected to actual Spaceflight for 6 days (Foton M3 satellite). Pthrp +/+, +/- and -/- osteoblasts were also exposed to simulated microgravity for periods varying from 6 days to 6 weeks. While COs displayed little change in viability in 0g, viability of all TOs rapidly decreased in inverse proportion to PTHrP expression levels. Furthermore, Pthrp+/+ TOs displayed a sharp viability decline after 2 weeks at 0g. Microarray analysis of Pthrp+/+ TOs after 6 days in simulated 0g revealed expression changes in genes encoding prolactins, apoptosis/survival molecules, bone metabolism and extra-cellular matrix composition proteins, chemokines, insulin-like growth factor family members and Wnt-related signalling molecules. 88% of 0g-induced expression changes in Pthrp+/+ cells overlapped those caused by Pthrp ablation in normal gravity, and pulsatile treatment with PTHrP1-36 not only reversed a large proportion of 0g-induced effects in Pthrp+/+ TOs but maintained viability over 6-week exposure to microgravity. Our results confirm PTHrP efficacy as an anabolic agent to prevent microgravity-induced cell death in TOs.

Histonefection: Novel and potent non-viral gene delivery.

Protein/peptide-mediated gene delivery has recently emerged as a powerful approach in non-viral gene transfer. In previous studies, we and other groups found that histones efficiently mediate gene transfer (histonefection). Histonefection has been demonstrated to be effective with various members of the histone family. The DNA binding domains and natural nuclear localisation signal sequences make histones excellent candidates for effective gene transfer. In addition, their positive charge promotes binding to anionic molecules and helps them to overcome the negative charge of cells that is an important barrier to cellular penetration. Histonefection appears to have particular promise in cancer gene transfer and therapy.

Ahi-1, a novel gene encoding a modular protein with WD40-repeat and SH3 domains, is targeted by the Ahi-1 and Mis-2 provirus integrations.

The Ahi-1 locus was initially identified as a common helper provirus integration site in Abelson pre-B-cell lymphomas and shown to be closely linked to the c-myb proto-oncogene. Since no significant alteration of c-myb expression was found in Abelson murine leukemia virus-induced pre-B-lymphomas harboring a provirus inserted within the Ahi-1 locus, this suggested that it harbors another gene whose dysregulation is involved in tumor formation. Here we report the identification of a novel gene (Ahi-1) targeted by these provirus insertional mutations and the cloning of its cDNA. The Ahi-1 proviral insertions were found at the 3' end of the gene, in an inverse transcriptional orientation, with most of them located around and downstream of the last exon, whereas another insertion was within intron 22. In addition, another previously identified provirus insertion site, Mis-2, was found to map within the 16th intron of the Ahi-1 gene. The Ahi-1 cDNA encodes a 1,047-amino-acid protein. The predicted Ahi-1 protein is a modular protein that contains one SH3 motif and seven WD40 repeats. The Ahi-1 gene is conserved in mammals and encodes two major RNA species of 5 and 4.2 kb and several other shorter splicing variants. The Ahi-1 gene is expressed in mouse embryos and in several organs of the mouse and rat, notably at high levels in the brain and testes. In tumor cells harboring insertional mutations in Ahi-1, truncated Ahi-1/viral fused transcripts were identified, including some splicing variants with deletion of the SH3 domain. Therefore, Ahi-1 is a novel gene targeted by provirus insertion and encoding a protein that exhibits several features of a signaling molecule. Thus, Ahi-1 may play an important role in signal transduction in normal cells and may be involved in tumor development, possibly in cooperation with other oncogenes (such as v-abl and c-myc) or with a tumor suppressor gene (Nf1), since Ahi-1 insertion sites were identified in tumors harboring v-abl defective retroviruses or a c-myc transgene or in tumors exhibiting deletion of Nf1.

Role of endocytosis in the internalization of spermidine-C(2)-BODIPY, a highly fluorescent probe of polyamine transport.

The mechanism of transmembrane polyamine internalization in mammalian cells remains unknown. A novel fluorescent spermidine conjugate [Spd-C(2)-BODIPY; N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)-N'-(S -[spermidine-(N(4)-ethyl)]thioacetyl)ethylenediamine] was synthesized from N(4)-(mercaptoethyl)spermidine by a simple, one-step coupling procedure. In Chinese-hamster ovary (CHO) cells, Spd-C(2)-BODIPY accumulation was inhibited by exogenous putrescine, spermidine and spermine, was subject to feedback transport inhibition and was up-regulated by prior polyamine depletion achieved with a biosynthetic inhibitor. Probe internalization was decreased by about 85% in a polyamine-transport-deficient CHO mutant cell line. Using confocal laser scanning fluorescence microscopy, internalized Spd-C(2)-BODIPY was concentrated in vesicle-like structures similar to the recycling endosomes observed with fluorescent transferrin, which partly co-localized with the polyamine probe. In yeast, Spd-C(2)-BODIPY uptake was stringently dependent on receptor-mediated endocytosis, as determined with a mutant defective in early- endosome formation. On the other hand, Spd-C(2)-BODIPY did not mimic the substrate behaviour of natural polyamines in yeast, as shown by the lack of correlation of its uptake characteristics with the phenotypes of mutants defective in either polyamine transport or biosynthesis. These data suggest that endocytosis might be an integral part of the mechanism of polyamine transport in mammalian cells, and that the mammalian and yeast transport systems use qualitatively different transport mechanisms. However, the current data do not rule out the possibility that sequestration of the probe into vesicular structures might be secondary to its prior uptake via a "classical" plasma membrane carrier. Spd-C(2)-BODIPY, a highly sensitive probe of polyamine transport with biochemical parameters qualitatively similar to those of natural polyamines in mammalian cells, should be very useful for dissecting the pathway responsible for polyamine internalization.