High Throughput Screening of Biologically Functional Small Molecules for Modulating the Expression of FGFR1OP2/wit3.0 in Fibroblasts

Objectives: In skin wounds, inflammation associated cytokines and growth factors induce fibroblasts to undergo transdifferentiation allowing them to contract the wound. Though wounds in the oral cavity heal with a lesser degree of inflammation, they contract at a faster pace and exhibit reduced scarring compared to the skin. FGFR1OP2/wit3.0 is a small cytoplasmic peptide highly expressed by oral wound fibroblasts, but not by skin wound fibroblasts. In this study we aim to determine the exogenous factors possibly inducing FGFR1OP2/wit3.0 expression in skin fibroblasts through the development of a high throughput screening assay. Methods: This study examined the use of direct mRNA measurement assays for use in high throughput applications to find chemical agents that up or downregulate FGFR1OP2/wit3.0 expression. Using mouse clonal fibroblastic cell lines we characterized genetic expression, phenotype, and si-RNA knockdown of wit3.0. We then developed and optimized a protocol for high throughput screening on a bDNA platform. A pilot screening was performed at the UCLA MSSR. Results: Confocal microscopy revealed that the cytoskeletal arrangement of actin and wit3.0 were phenotypically different in the heterozygous knockout stem cell derived fibroblasts. wit3.0 (+/-) cells exhibited disorganized actin fibers, while the wildtype showed a more organized archetype. We found that wit3.0 was expressed in both L-929 and C3H/10T1/2 and appears in immunostaining as a cytoplasmic, granular protein. We were able to successfully perform a high throughput screening of 320 chemicals as a pilot study, and identified 7 compounds as modulators of wit3.0. Conclusion: This pilot study has identified biologically functional chemical compounds which could provide a novel clue in elucidating the mechanism of fibroblast-driven wound closure, and presents a new therapeutic potential in the field of wound healing