Initiation of early osteoblast differentiation events through the direct transcriptional regulation of Msx2 by FOXC1.

Hierarchal transcriptional regulatory networks function to control the correct spatiotemporal patterning of the mammalian skeletal system. One such factor, the forkhead box transcription factor FOXC1 is necessary for the correct formation of the axial and craniofacial skeleton. Previous studies have demonstrated that the frontal and parietal bones of the skull fail to develop in mice deficient for Foxc1. Furthermore expression of the Msx2 homeobox gene, an essential regulator of calvarial bone development is absent in the skull mesenchymal progenitors of Foxc1 mutant mice. Thus we sought to determine whether Msx2 was a direct target of FOXC1 transcriptional regulation. Here, we demonstrate that elevated expression of FOXC1 can increase endogenous Msx2 mRNA levels. Chromatin immunoprecipitation experiments reveal that FOXC1 occupies a conserved element in the MSX2 promoter. Using a luciferase reporter assay, we demonstrate that FOXC1 can stimulate the activity of the both human and mouse MSX2 promoters. We also report that reducing FOXC1 levels by RNA interference leads to a decrease in MSX2 expression. Finally, we demonstrate that heterologous expression of Foxc1 in C2C12 cells results in elevated alkaline phosphatase activity and increased expression of Runx2 and Msx2. These data indicate that Foxc1 expression leads to a similar enhanced osteogenic differentiation phenotype as observed with Msx2 overexpression. Together these findings suggest that a Foxc1->Msx2 regulatory network functions in the initial stages of osteoblast differentiation.

Genetic screening for synthetic lethal partners of polynucleotide kinase/phosphatase: potential for targeting SHP-1-depleted cancers.

A genetic screen using a library of 6,961 siRNAs led to the identification of SHP-1 (PTPN6), a tumor suppressor frequently mutated in malignant lymphomas, leukemias, and prostate cancer, as a potential synthetic lethal partner of the DNA repair protein polynucleotide kinase/phosphatase (PNKP). After confirming the partnership with SHP-1, we observed that codepletion of PNKP and SHP-1 induced apoptosis. A T-cell lymphoma cell line that is SHP-1 deficient (Karpas 299) was shown to be sensitive to a chemical inhibitor of PNKP, but resistance was restored by expression of wild-type SHP-1 in these cells. We determined that while SHP-1 depletion does not significantly impact DNA strand-break repair, it does amplify the level of reactive oxygen species (ROS) and elevate endogenous DNA damage. The ROS scavenger WR1065 afforded protection to SHP-1-depleted cells treated with the PNKP inhibitor. We propose that codisruption of SHP-1 and PNKP leads to an increase in DNA damage that escapes repair, resulting in the accumulation of cytotoxic double-strand breaks and induction of apoptosis. This supports an alternative paradigm for synthetic lethal partnerships that could be exploited therapeutically.