Loss of Foxc1 and Foxc2 function in chondroprogenitor cells disrupts endochondral ossification.

Endochondral ossification initiates the growth of the majority of the mammalian skeleton and is tightly controlled through gene regulatory networks. The forkhead box transcription factors Foxc1 and Foxc2 regulate aspects of osteoblast function in the formation of the skeleton, but their roles in chondrocytes to control endochondral ossification are less clear. Here, we demonstrate that Foxc1 expression is directly regulated by the activity of SRY (sex-determining region Y)-box 9, one of the earliest transcription factors to specify the chondrocyte lineage. Moreover, we demonstrate that elevated expression of Foxc1 promotes chondrocyte differentiation in mouse embryonic stem cells and loss of Foxc1 function inhibits chondrogenesis in vitro. Using chondrocyte-targeted deletion of Foxc1 and Foxc2 in mice, we reveal a role for these factors in chondrocyte differentiation in vivo. Loss of both Foxc1 and Foxc2 caused a general skeletal dysplasia predominantly affecting the vertebral column. The long bones of the limbs were smaller, mineralization was reduced, and organization of the growth plate was disrupted; in particular, the stacked columnar organization of the proliferative chondrocyte layer was reduced in size and cell proliferation was decreased. Differential gene expression analysis indicated disrupted expression patterns of chondrogenesis and ossification genes throughout the entire process of endochondral ossification in chondrocyte-specific Foxc1/Foxc2 KO embryos. Our results suggest that Foxc1 and Foxc2 are required for normal chondrocyte differentiation and function, as loss of both genes results in disorganization of the growth plate, reduced chondrocyte proliferation, and delays in chondrocyte hypertrophy that prevents ossification of the skeleton.

PKC activator therapeutic for mild traumatic brain injury in mice.

Traumatic brain injury (TBI) is a frequent consequence of vehicle, sport and war related injuries. More than 90% of TBI patients suffer mild injury (mTBI). However, the pathologies underlying the disease are poorly understood and treatment modalities are limited. We report here that in mice, the potent PKC activator bryostatin1 protects against mTBI induced learning and memory deficits and reduction in pre-synaptic synaptophysin and post-synaptic spinophylin immunostaining. An effective treatment has to start within the first 8h after injury, and includes 5 × i.p. injections over a period of 14 days. The treatment is dose dependent. Exploring the effects of the repeated bryostatin1 treatment on the processing of the amyloid precursor protein, we found that the treatment induced an increase in the putative α-secretase ADAM10 and a reduction in ß-secretase activities. Both these effects could contribute towards a reduction in ß-amyloid production. These results suggest that bryostatin1 protects against mTBI cognitive and synaptic sequela by rescuing synapses, which is possibly mediated by an increase in ADAM10 and a decrease in BACE1 activity. Since bryostatin1 has already been extensively used in clinical trials as an anti-cancer drug, its potential as a remedy for the short- and long-term TBI sequelae is quite promising.

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.