The role of p21 in regulation of injury response in lung epithelial cells

Abstract

After lung injury epithelial cells are damaged may be shed thus denuding the basement membrane. The normal response to injury is usually an inflammatory reaction, deposition of new extracellular matrix that incorporates various growth factors, influx of inflammatory cells and regeneration of lost epithelial cells. Regeneration involves migration, proliferation and differentiation of stem cells to cover the exposed area. In the lung bronchiolar region Clara cells are thought to be the progenitor cells. Changes in cell cycle and behaviour of lung epithelial cells in several models of lung injury and in vitro primary culture were studied. The expression of the cell cycle control genes, p21, p53, PCNA and p27, in mouse models of three important human diseases mainly fibrosis, asthma and Mycobacterium tuberculosis infection were investigated. p21 expression by epithelial cells increased in fibrosis, decreased in asthma but did not change after TB infection. Clara cells were isolated from mice and cultured in serum free conditions for up to five days. They were characterised in terms of proliferation (BrdU, PCNA & mitosis), differentiation (lectins, integrins, cytokeratins & CC10), death (apoptosis and necrosis) and cell cycle regulation (p21, p53, EGFR & p27). The effects of cell matrix interactions on Clara cell proliferation, differentiation, cell cycle control and death were studied by varying the extracellular matrix composition and disruption of cell-integrin interactions in both wildtype and p21 knockout mice. Matrix changes did not influence significantly the proliferation, differentiation, death and cell cycle regulation in Clara cell cultures in both wildtype and p21 knockout mice, except when laminin was present. Significant differences in cytokeratin expression, cell cycle regulation and death pathways were observed in wildtype mice when compared to p21 knockouts. Some of the differences include lower expression of cytokeratin 8 and 19 in p21 ko mice, lower cytoplasmic but higher nuclear PCNA expression in p21 ko mice, a lower necrotic rate in p21 ko mice, lower cytoplasmic but higher nuclear p53 expression in p21 ko mice. By using a beta-1 blocking antibody the cellintegrin disruption was carried. Significant differences were observed upon beta-1 integrin blocking including an increase in cytokeratin 8 and 19 expression in p21 ko mice, decrease in proliferation in both wt and p21 ko mice, an increase in cytoplasmic PCNA in both wt and p21 ko mice, an increase in apoptosis rate in both wt and p21 mice, increase in nuclear p21 in wt mice, an increase in nuclear p53 in both wt and p21 ko mice, and a decrease in cytoplasmic but an increase in nuclear p27. The two forms of p21 were studied and it was found that the cytoplasmic p21 is a C-terminal truncated protein with a weight of 17 kDa. The complex of p21 in both the cytoplasm and nucleus were studied. The nuclear p21 was found to form complexes with PCNA, cdk2, cdk4, cdk6, cyclin D3 and cyclin E, while the cytoplasmic p21 form complexes with cdk4 and cyclin D3. In conclusion, the work presented in this thesis demonstrates that p21 is an important factor for the proliferation, differentiation and death of Clara cells. It also demonstrates that cell-matrix interactions form an important part in the regulation of Clara cells. Two forms of p21, a cytoplasmic and a nuclear protein are identified and their importance in the regulation and division of Clara cells is discussed.