The effects of mechanically loaded osteocytes and inflammation on bone remodeling in a bisphosphonate-induced environment.

Bisphosphonate-related osteonecrosis of the jaw is a disease appearing after tooth removal in patients undergoing bisphosphonate treatment for metastasizing cancers and osteoporosis. The complexity of the condition requires a multicellular model to address the net effects of two key risk factors: mechanical trauma (pathologic overload) and inflammation. In this work, a system comprised of a polydimethylsiloxane chip and mechanical loading device is used to expose bisphosphonate-treated osteocytes to mechanical trauma. Specifically, osteocytes are treated with the potent nitrogen-containing bisphosphonate, zoledronic acid, and exposed to short-term pathologic overload via substrate stretch. During bone remodeling, osteocyte apoptosis plays a role in attracting pre-osteoclasts to sites of damage; as such, lactate dehydrogenase activity, cell death and protein expression are evaluated as functions of load. Additionally, the effects of osteocyte soluble factors on osteoclast and osteoblast functional activity are quantified. Osteoclast activity and bone resorption are quantified in the presence and absence of inflammatory components, lipopolysaccharide and interferon gamma. Results suggest that inflammation associated with bacterial infection may hinder bone resorption by osteoclasts. In addition, osteocytes may respond to overload by altering expression of soluble signals that act on osteoblasts to attenuate bone formation. These findings give insight into the multicellular interactions implicated in bisphosphonate-related osteonecrosis of the jaw.

New negative feedback regulators of Egfr signaling in Drosophila.

The highly conserved epidermal growth factor receptor (Egfr) pathway is required in all animals for normal development and homeostasis; consequently, aberrant Egfr signaling is implicated in a number of diseases. Genetic analysis of Drosophila melanogaster Egfr has contributed significantly to understanding this conserved pathway and led to the discovery of new components and targets. Here we used microarray analysis of third instar wing discs, in which Egfr signaling was perturbed, to identify new Egfr-responsive genes. Upregulated transcripts included five known targets, suggesting the approach was valid. We investigated the function of 29 previously uncharacterized genes, which had pronounced responses. The Egfr pathway is important for wing-vein patterning and using reverse genetic analysis we identified five genes that showed venation defects. Three of these genes are expressed in vein primordia and all showed transcriptional changes in response to altered Egfr activity consistent with being targets of the pathway. Genetic interactions with Egfr further linked two of the genes, Sulfated (Sulf1), an endosulfatase gene, and CG4096, an A Disintegrin And Metalloproteinase with ThromboSpondin motifs (ADAMTS) gene, to the pathway. Sulf1 showed a strong genetic interaction with the neuregulin-like ligand vein (vn) and may influence binding of Vn to heparan-sulfated proteoglycans (HSPGs). How Drosophila Egfr activity is modulated by CG4096 is unknown, but interestingly vertebrate EGF ligands are regulated by a related ADAMTS protein. We suggest Sulf1 and CG4096 are negative feedback regulators of Egfr signaling that function in the extracellular space to influence ligand activity.

Efficient genetic method for establishing Drosophila cell lines unlocks the potential to create lines of specific genotypes.

Analysis of cells in culture has made substantial contributions to biological research. The versatility and scale of in vitro manipulation and new applications such as high-throughput gene silencing screens ensure the continued importance of cell-culture studies. In comparison to mammalian systems, Drosophila cell culture is underdeveloped, primarily because there is no general genetic method for deriving new cell lines. Here we found expression of the conserved oncogene Ras(V12) (a constitutively activated form of Ras) profoundly influences the development of primary cultures derived from embryos. The cultures become confluent in about three weeks and can be passaged with great success. The lines have undergone more than 90 population doublings and therefore constitute continuous cell lines. Most lines are composed of spindle-shaped cells of mesodermal type. We tested the use of the method for deriving Drosophila cell lines of a specific genotype by establishing cultures from embryos in which the warts (wts) tumor suppressor gene was targeted. We successfully created several cell lines and found that these differ from controls because they are primarily polyploid. This phenotype likely reflects the known role for the mammalian wts counterparts in the tetraploidy checkpoint. We conclude that expression of Ras(V12) is a powerful genetic mechanism to promote proliferation in Drosophila primary culture cells and serves as an efficient means to generate continuous cell lines of a given genotype.