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Elucidation of the chondrogenic program using a combination of biology and technology Garcha, Kamal

Abstract

Skeletogenesis is associated with the aggregation of mesenchymal cells into condensations that prefigure the mature skeleton under the influence of numerous signaling molecules. In the developing murine limb, Fibroblast growth factors (FGFs) (principally 4 and 8) are expressed in the apical ectodermal ridge (AER) and signal to the underlying distal mesenchymal cells. In this manner, FGFs are thought to influence the proliferation of chondroprogenitors, thereby modulating the growth of pre-cartilage condensations. To study these effects the chondrogenic program can be effectively modeled in vitro using high-density cultures of primary cells isolated from the murine limb bud (El 1.5). Treatment of distal mesenchymal cultures with FGF4 leads to a transient increase in proliferation and expansion of prechondrogenic condensations. Using transcriptional profiling with DNA microarrays we observed that consistent with the changes observed in cell proliferation, FGF4 treatment resulted in an ∼ 18 fold increase in the expression of Cdkn2b — a cyclin dependent kinase inhibitor. SiRNA knockdown of Cdkn2b resulted in sustained mesenchymal proliferation. Further, we determined that FGF4 regulates Cdkn2b through a MEKI/ERK-dependent pathway. Additionally, we show that FGF4 promotes cell survival through the regulation of NFiB via upregulation of its activator RIPK4. To enhance our studies of chondrogenesis, a high throughput reporter gene based assay was developed. Using this technology we performed a chemical genetic screen of ∼1500 chemical compounds to assess their ability to regulate reporter gene activity. Of these compounds, 28 yielded a >2.5 fold increase in S0X5/6/9 reporter gene activity. Secondary histological screens confirmed increased cartilage formation in response to treatment of primary limb cultures with several of these newly identified chemical enhancers of chondrogenesis. Through bioinformatic analysis of microarray data in comparison with the known targets of the screened chemical compounds, we have shown that inhibition of potassium channel KCND2 (Kv4.2) promotes chondrogenesis. Additionally, it was found that prochondrogenic bone morphogenetic protein 4 (BMP4) downregulated the expression of Kcnd2. These data have revealed an unanticipated role of potassium channels during chondrogenesis. Thus by using a combined approach of microarray analysis and chemical genetics we have further characterized the intricacies of the chondrogenic program.

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