Summary: Much is known regarding the role of Indian hedgehog (Ihh) in endochondral ossification, where Ihh regulates multiple steps of chondrocyte differentiation. The Ihh 2/2 phenotype is most notable for severely foreshortened limbs and a complete absence of mature osteoblasts. A far less explored phenotype in the Ihh 2/2 mutant is found in the calvaria, where bones form predominately through intramembranous ossification. We investigated the role of Ihh in calvarial bone ossification, finding that proliferation was largely unaffected. Instead, our results indicate that Ihh is a pro-osteogenic factor that positively regulates intramembranous ossification. We confirmed through histologic and quantitative gene analysis that loss of Ihh results in reduction of cranial bone size and all markers of osteodifferentiation. Moreover, in vitro studies suggest that Ihh loss reduces Bmp expression within the calvaria, an observation that may underlie the Ihh 2/2 calvarial phenotype. In conjunction with the newly recognized roles of Hedgehog deregulation in craniosynostosis, our study defines Ihh as an important positive regulator of cranial bone ossification.

Introduction Cleft lip and palate are common birth defects and represent a large biomedical burden. Through the use of animal models, the molecular underpinnings of cleft palate are becoming increasingly clear. Indian Hedgheg (Ihh) has been shown to be associated craniofacial development and active in the palatine bone. We hypothesize that Ihh activity plays a role in osteogenesis within the secondary palate and that defects in this pathway may inhibit the osteogenesis of the secondary palate. Methods Palates were isolated from wild type mice during the period of palate development (e9.5-e17.5). qRT-PCR was used for detecting gene expression during osteogenic differentiation and cellular differentiation (Shh, Ihh, Ptc1, Gli1, Gli2, Gli3, Runx2, Alp, Col1a1). Next, palates were analyzed by H&E, Aniline Blue, Pentachrome, and In situ Hybridization to assess osteogenesis of the palatal shelf and expression of hedgehog pathway genes. Finally, the palate of Indian Hedgehog null mice was analyzed to determine the effect of genetic deficiency on palatal development osteogenesis. Results Increased Indian Hedgehog and osteogenic signaling coincided with ossification and fusion of the palate in wild-type mice. This included a 5-150 fold peak in expression of Hedgehog elements, including Ihh, at e15.5 as compared to e9.5. Contrarily, loss of Indian Hedgehog by genetic knockout (Ihh −/−) resulted in decreased secondary palate ossification. Conclusion Our results suggest a role for Hedgehog signaling during palatal ossification. The hedgehog pathway is activated during palatal fusion and deletion of Indian hedgehog leads to diminished ossification of the secondary hard palate.

Bone regeneration is a complex event that requires the interaction of numerous growth factors. Fibroblast growth factor (Fgf)-ligands have been previously described for their importance in osteogenesis during development. In the current study, we investigated the role of Fgf-18 during bone regeneration. By utilizing a unicortical tibial defect model, we revealed that mice haploinsufficient for Fgf-18 have a markedly reduced healing capacity as compared with wild-type mice. Reduced levels of Runx2 and Osteocalcin but not Vegfa accompanied the impaired bone regeneration. Interestingly, our data indicated that upon injury angiogenesis was not impaired in Fgf-18 + / -mice. Moreover, other Fgf-ligands and Bmp-2 could not compensate for the loss of Fgf-18. Finally, application of FGF-18 protein was able to rescue the impaired healing in Fgf-18 + / -mice. Thus, we identified Fgf-18 as an important mediator of bone regeneration, which is required during later stages of bone regeneration. This study provides hints on how to engineering efficiently programmed bony tissue for long bone repair.

The primary cilium is an organelle that senses cues in a cell’s local environment. Some of these cues constitute molecular signals; here, we investigate the extent to which primary cilia can also sense mechanical stimuli. We used a conditional approach to delete Kif3a in pre-osteoblasts and then employed a motion device that generated a spatial distribution of strain around an intra-osseous implant positioned in the mouse tibia. We correlated interfacial strain fields with cell behaviors ranging from proliferation through all stages of osteogenic differentiation. We found that peri-implant cells in the Col1Cre;Kif3afl/fl mice were unable to proliferate in response to a mechanical stimulus, failed to deposit and then orient collagen fibers to the strain fields caused by implant displacement, and failed to differentiate into bone-forming osteoblasts. Collectively, these data demonstrate that the lack of a functioning primary cilium blunts the normal response of a cell to a defined mechanical stimulus. The ability to manipulate the genetic background of peri-implant cells within the context of a whole, living tissue provides a rare opportunity to explore mechanotransduction from a multi-scale perspective.

PURPOSE: Hedgehog (HH) pathway has been implicated in maintenace and survival of breast cancer and cancer stem cells, EMT regulation, breast carcinogenesis and resistance to chemoradiation in solid tumors. Dysregulation of this pathway occurs in more than 50% of breast cancers. We have already demonstrated that immune-induced epithelial to mesenchymal transition (EMT) leads to the generation of breast cancer stem cells (BCSCs) from a parental epithelial breast cancer cell line in an in vivo murine model (Cancer Research 2009). The aim of our study is to demonstrate that resistance in the BCSCs is linked to HH hyperactivation and that the selective blockade of this pathway could eradicate this subpopulation of BCSCs in preclinical models. METHODS: One parental epithelial (E) and three mesenchymal BCSCs cell lines (M1, M2 and M3) were used for all the in vitro experiments. qRT-PCR was used to quantify the expression of the gene components of the pathway and gene targets. Measurement of Gli1, caspase 3 and Cyclin D1 protein expression was determined by Western blot. Cell viability assays were performed with Paclitaxel and the HH inhibitor GDC-0449. Apoptosis assays were performed when cell lines were treated with paclitaxel (10nM), GDC-0449 (25uM) or the combination of them. For the in vivo xenografted mouse model, 106 cells (E and M2) were inoculated sc respectively into the flank of 6–8 week-old syngeneic female FVB/N-TgN (MMTVneu) 202Mul/J mice. Treatment was started on daily GDC-0449 (ip), paclitaxel (ip on days 1, 4 and 8) or both drugs when tumors reached 0.5 cm2. Statistical analysis was performed using one-way ANOVA with the Bonferroni multiple comparison test. RESULTS: Activity of the HH measured by the expression of Gli1 was increased in BCSC cell lines as compared to E cell line (p < 0.001). This was antagonized as expected by treatment with GDC-0449. Reporter and expression assay showed that GDC-0449 decreases Gli1 transcriptional activity and levels of a HH downstream target gene, cyclin D1. In all BCSC lines (but not in the E cell line), a synergistic effect was showed with the combination treatment as compared to control (p ≤ 0.01) and to paclitaxel or GDC-0449 alone (p ≤ 0.05 respectively) as well as a highly increased apoptotic activity (p ≤ 0.001 with control and p ≤ 0.05 with monotherapy). Tumors derived from M2 injection regressed when the combination schedule was administered (p ≤ 0.05) at 2 weeks after the start of the treatment. CONCLUSION: The inhibition of the HH pathway with GDC-0449 plus paclitaxel demonstrates a synergistic therapeutic effect in comparison to monotherapy, as shown by increased apoptotic activity, cell cycle arrest and significant reduction of tumor size in vivo. Together these results provide the rationale for future clinical trials including the blockade HH and standard chemotherapy in order to eradicate the whole population of tumor cells (also BCSCs) within the tumors and avoid disease relapse and metastasis in breast cancer patients. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-03-07.