Cell growth and differentiation are usually antagonistic. Proteins of the basic helix-loop-helix (bHLH) family bind DNA and play important roles in the differentiation of specific cell types. Id proteins heterodimerize with bHLH transcription factors, blocking their activation of lineage-specific gene expression and thereby inhibiting cellular differentiation. To examine the effect of Id-2 on cell proliferation, we overexpressed Id-2 in the human osteosarcoma cell line U2OS. Id-2 expression in U2OS reduced the serum requirement for growth and stimulated cellular proliferation by shortening the doubling time and increasing the percentage of cells in S phase. We demonstrated that Id-2 expression was able to reverse the inhibition of cellular proliferation and the block in cell cycle progression mediated by the product of the retinoblastoma tumor suppressor gene pRB. This effect was not associated with changes in the state of pRb phosphorylation in transfected cells. In vitro, unphosphorylated pRb from cell lysates specifically bound Id-2 but was not able to bind a mutated form of Id-2 lacking the HLH domain that also did not antagonize the growth arrest by pRb. In vitro-synthesized pRb containing mutations within the E1A/large T-binding pocket did not bind Id-2. However, wild-type pRb was able to bind to a region of Id-2 corresponding to only the HLH domain. In vivo, a physical association between Id-2 and pRb was seen in cross-linked extracts from SAOS-2 cells transfected with Id-2 and pRb. Our data identify a role for Id-2 in the regulation of cellular proliferation and suggest that the interaction between Id-2 and pRb is a molecular pathway over which synchronous changes in growth and differentiation are mediated in vivo.
Chronic tympanic membrane (TM) perforations can cause otorrhea. To date, various types of tissue engineering techniques have been applied for the regeneration of chronic TM perforations. However, the application of nanofibers with radially aligned nanostructures and the simultaneous release of growth factors have never been applied in the regeneration of chronic TM perforations. Here, epidermal growth factor (EGF)–releasing radially aligned nanofibrous patches (ERA‐NFPs) are developed and applied for the regeneration of chronic perforated TMs. First, radial alignments and the presence of EGF in the ERA‐NFPs are analyzed. EGF is confirmed to be released from the ERA‐NFPs until 8 weeks. In an in vitro study, cell viability assay, immunocytochemistry, and wound‐healing assay indicate rational enhancement of healing by the combination of radial alignments and EGF release. The effect of ERA‐NFPs on TM cells is revealed by quantitative real‐time polymerase chain reaction. An in vivo animal study shows that the ERA‐NFPs effectively stimulates the healing of the chronic TM perforations. The TMs healed by ERA‐NFPs show histological properties similar to those of normal TMs. These results indicate that ERA‐NFPs may be an efficient platform for the regeneration of chronic TM perforations, laying the foundation for nonsurgical treatments of chronic otitis media.
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